CZ2000681A3 - Oligonucleotides allowing the identification of precursors of amidated polypeptide hormones - Google Patents

Abstract

Oligonucleotides and their use as probes for identifying mRNA encoding precursors of amidated polypeptide hormones, thereby identifying novel amidated polypeptide hormones. Oligonucleotides with a disclosed nucleotide sequence and a method for identifying precursors of amidated hormones.

Description

The present invention relates to novel oligonucleotides and their use as probes for identifying mRNA encoding precursors of amidated polypeptide hormones and for identifying novel amidated polypeptide hormones. The invention therefore relates to oligonucleotides having the nucleotide sequence described below and to a method for identifying precursors of hormones.

State of the art

Amidated polypeptide hormones are synthesized in the form of precursors that undergo maturation. This maturation consists of an amidation reaction.

The C-terminal amidation reaction is a characteristic reaction of amidated polypeptide hormones. This reaction, which occurs on the precursors of one or more hormones, allows the maturation of the hormone and also ensures its biostability in the physiological medium: the amide group formed is less vulnerable than the free acid function. The hormone is therefore more resistant to carboxypeptidases, remains active in the cell for a longer period and retains optimal affinity for its receptor site.

Amidation has been extensively described (Peptide amidation, Alan F. Bradbury and Derek G. Smyth, TIBS .16.: 112 to 115, March 1991 Functional and structural characterisation of peptidylamidoglyco1 ate lyase, the enzyme catalysing the second step in peptide amidation, A.G. Katopodis, D.S. Ping, C.E. Smith

and S.W.May, Biochemistry, 30 (25): 6189-6194, June 1991), and its mechanism is as follows:

~ cleavage of the hormone polypeptide chain precursor by an endoprotease at two basic amino acids, i.e. arginine and/or lysine,

- then two cleavages by carboxypeptidase occur, leading to an extended glycine intermediate, ~ the enzyme PAM (peptidylglycine-α-amidating monooxygenase) contains two distinct enzymatic activities: first, it converts the extended glycine intermediate into an α-hydroxyglycine derivative, the subunit of the PAM enzyme that performs the action is PHM (peptidylglycine-α-hydroxylating monooxygenase). The derivative obtained serves as a substrate for the second subunit of the PAM (designated PAL: peptidylglycine-α-amidating lyase), which fixes the amino function of glycine to an amino acid, immediately adjacent to the N-terminal site and releases glyoxylate.

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

It is known that amidated polypeptide hormones intended for secretion outside the endoplasmic reticulum contain a consensus signal sequence of approximately fifteen to thirty amino acids, which sequence is present at the N-terminal end of the polypeptide chain. It is later cleaved by the signal peptidase enzyme, so that it is no longer present in the protein once secreted (see F. Cuttitta, The Anatomical Record, .236, 87-93 (1993) and references cited therein).

Finding a new protein is not easy at present. Proteins can be isolated and purified by various techniques: precipitation at the isoelectric point, selective extraction with certain solvents and then purification by crystallization, countercurrent distribution, adsorption, partition or ion exchange chromatography, electrophoresis, etc. However, these techniques presuppose knowledge of the properties of the protein to be isolated. Moreover, if a pure sample of the new protein in question is available at therapeutic levels, there are still several steps to go before a genetically modified microorganism capable of synthesizing it is available.

The method proposed by the present invention offers the advantage of using the characteristic peptide sequence of the precursor of all hitherto known amidated hormones, allowing the simultaneous detection of several new hormones of this category. This screening is carried out by direct identification of the nucleotide sequence encoding said precursors in cDNA libraries prepared from tissues in which the precursors of these hormones can be synthesized.

Research using this method is much less restrictive than the commonly used biochemical techniques mentioned above because:

- can lead to the isolation of several different precursors in the same tissue based on the same principle,

- allows the detection of precursors corresponding to hormones, having very different biochemical and biological properties under the same technical conditions, ~ allows the concomitant identification of all peptide hormones that may be contained in the same precursor.

The result of this invention is that it allows for a not inconsiderable saving of time and money in an area where research and development costs represent a very high proportion of turnover.

The present invention will also allow the pharmacological study of active substances having a fundamental physiological role in the mammalian organism: hormones and in particular amidated polypeptide neurohormones. Since the cDNA corresponding to the active substances is available for the first time, it will then be possible to introduce a cloned vector by genetic engineering, which will lead to the synthesis of hormones having therapeutic use by means of microorganisms.

The essence of the invention

The invention relates first to a single-stranded oligonucleotide OX which can hybridize under mild conditions to an oligonucleotide OY of the sequence Y1 ~Y2--Y3~-Y4~Y5, where Y1 represents a nucleotide sequence of 1 to 12 nucleotides or Y1 is suppressed, Y2 represents a trinucleotide that encodes Gly, Y3 and Y4 independently represent a trinucleotide that encodes Arg or Lys and Y5 represents a nucleotide sequence of 1 to 20 nucleotides or Y5 is suppressed.

The term nucleotide refers to the monomeric unit of RNA

or DNA having the chemical structure of a phosphate ester of a nucleoside. A nucleoside is formed by the bonding of a purine base (purine, adenine, guanine, or analogs) or a pyrimidine base (pyrimidine, cytosine, uracil, or analogs) to ribose or deoxyribose. An igonucleotide is a polymer of nucleotides that designates a primer sequence, probe, or fragment of RNA or DNA.

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

Preferably, OX can hybridize with OY under stringent conditions.

More preferably, hybridization of OX with the OY oligonucleotide of the sequence Y2-Y3-Y4-Y5 is possible.

Even more preferred is the possible hybridization of OX with the OY nucleotide sequence Y1-Y2-Y3-Y4 or Y2-Y3-Y4.

In a particular case, OX may hybridize to oligonucleotide OY such that Y5 represents the nucleotide sequence Y6-Y7-Y8-Y9, in which Y6 represents a trinucleotide that codes for Ser, Thr or Tyr, Y7 represents a trinucleotide that codes for any amino acid, Y8 represents a trinucleotide that codes for Glu or Asp and Y9 represents a nucleotide sequence containing 1 to 12 nucleotides. In a very particular case, OX may hybridize to oligonucleotide OY such that Y1 and Y9 are suppressed.

In a very special case, OX can hybridize with the oligonucleotide OY, in which Y2 represents a trinucleotide that codes for Gly, Y3 represents a trinucleotide that codes for Lys, Y4 represents a trinucleotide that codes for Arg, and Y5 represents a sequence of 3 nucleotides that codes for Ser—A1 and ~G1u.

This sequence was determined using a statistical study of 27 known amidation sites and led to the definition of a given amino acid pattern at 6 positions: G1y~Lys~Arg-Ser-Ala~G1u.

Due to the degeneracy of the genetic code and the high number of codons corresponding to Gly (4 codons), Arg (6 codons) and Ser (6 codons), the 1-nucleotide sequence was constructed using two procedures that allow this degeneracy to be taken into account:

- the use of certain positions of inosine, which is a nucleotide in which the nitrogen base of hypoxanthine binds in a random manner with the 4 nitrogen bases that complement the DNA, ~ the change in the nature of the nitrogen base incorporated at certain positions, thereby creating a series of combinations of 1 igonucleotides proportional to the number of different bases introduced.

The present invention also relates to an oligonucleotide OY comprising 9 to 42 nucleotides of the sequence Y1 "Y2~-Y3~Y4"Y5, where Y1 represents a nucleotide sequence of 1 to 12 nucleotides or Y1 is suppressed, Y2 represents a trinucleotide that codes for Gly, Y3 and Y4 independently represent a trinucleotide that codes for Arg or Lys and Y5 represents a nucleotide sequence of 1 to 21 nucleotides or Y5 is suppressed.

The invention preferably relates to the oligonucleotide OY such that Y1 is suppressed or such that Y5 is suppressed.

The invention particularly relates to the oligonucleotide OY such that Y5 represents the nucleotide sequence Y6-Y7-Y8-Y9, in which Y6 represents a trinucleotide that codes for Ser, Thr or Tyr, Y7 represents a trinucleotide that codes for any amino acid, Y8 represents a trinucleotide that codes for Glu or Asp and Y9 represents a nucleotide sequence containing 1 to 12 nucleotides.

The invention particularly relates to the oligonucleotide OY such that Y1 and Y9 are suppressed.

In a very particular case, the invention relates to an oligonucleotide OY characterized in that Y1 is suppressed, Y2 represents a trinucleotide that codes for Gly, Y3 represents a trinucleotide that codes for Lys, Y4 represents a trinucleotide that codes for Arg and Y5 represents a sequence of three trinucleotides that codes for

Ser-A1 and —G1u.

The present invention also relates to a single-stranded oligonucleotide O2 characterized in that it contains 15 to 39 nucleotides and is capable of hybridizing with a consensus signal sequence characteristic of amidated polypeptide hormones, said sequence having the formula 21-22-Z3-24-2526-27, in which 21 represents a nucleotide sequence of 1 to 12 nucleotides or 21 is suppressed, 22 and 23 represent two trinucleotides that code for Leu, 24 and 25 represent two trinucleotides that code for any two amino acids, 26 represents a trinucleotide that codes for Leu and 27 represents a nucleotide sequence of 1 to 12 nucleotides or is suppressed.

In this invention, the term hormone will be understood to mean amidated polypeptide hormones of the endocrine system and in particular neurohormones.

A consensus signal sequence is a sequence carried by protein precursors that are secreted by cells after their maturation.

Finally, the present invention relates to a group of oligonucleotides OX or 02 that form a combinatorial library.

In the present invention, the term combinatorial library is understood to mean a group of oligonucleotides synthesized in such a way that a nucleotide sequence is taken as a model, which codes for a sequence of amino acids, some of which may vary. Due to the degeneracy of the genetic code, a group of different oligonucleotides is obtained.

The invention also relates to a method for identifying a peptide precursor having an amidated C-terminal end, characterized by the following sequential steps:

1- Obtaining a DNA bank,

2™ Hybridization of one or more 1 igonucleotides using a given DNA bank,

3~ Identification of the DNA sequence or sequences of a given library that hybridizes with the OX oligonucleotide,

4™ Identification in this sequence or sequences of one or more peptides with a possible amidated C-terminus.

It will be preferred that the DNA bank is a cDNA bank.

Complementary DNA (cDNA) is a nucleotide chain whose sequence is complementary to the sequence of mRNA. The reaction leading to single-stranded cDNA is catalyzed by reverse transcriptase. Double-stranded cDNA can be obtained by the action of DNA polymerase, which is then inserted by ligase into a plasmid or vector derived from bacteriophage lambda.

A cDNA library contains cDNA corresponding to cytoplasmic mRNA extracted from a given cell. A cell is called complete if it contains at least one bacterial clone for each starting mRNA.

Hybridization occurs when two oligonucleotides have highly complementary nucleotide sequences and can associate along their length by forming hydrogen bonds between complementary bases.

Particularly preferred will be a method in which the oligonucleotide OX can be detected using a labeling agent such as ³² P or digoxigenin.

The reagents most commonly used for radiolabeling are β-ray emitting elements, such as ³ H, ¹² Q, ³² P, ³³ P, and ³ »S.

The labeling of oligonucleotides is carried out by introducing phosphate

group carried by (gamma- ³² P)-ATP at its 5' end, this reaction is catalyzed by the enzyme T4-polynucleotide kinase. Labeling with digoxigenin is immunoenzymatic, digoxigenin is attached to a nitrogen base and incorporated into the oligonucleotide. Its presence is detected using an antibody directed against digoxigenin and attached to alkaline phosphatase. The presence is detected using a color induced by the substrate hydrolyzed by alkaline phosphatase.

Other labeling techniques may be used: oligonucleotides chemically modified to contain a complex metal reagent (lanthanide complexes are often used), a biotin-containing group or an acridine ester, a fluorescent substance (fluorescein, rhodamine, Texas red), or others.

A method for identifying an amidated polypeptide hormone precursor that uses combinatorial libraries of OX oligonucleotides in the hybridization step will be particularly preferred.

The invention also relates to a method for identifying a peptide precursor having an amidated C-terminus, which consists of the following steps:

obtaining a DNA bank, using the PCR technique to amplify the fragment of interest using a group of oligonucleotides OX and another group of oligonucleotides OZ, identifying the DNA sequence of the given bank that hybridizes with the OX oligonucleotide and that was amplified by the PCR reaction,

1

identification in this sequence of one or more peptides with a possible amidated C-terminus.

The term fragment of interest refers to a cDNA sequence that encodes a precursor of one or more amidated polypeptide hormones.

The PCR (polymerase chain reaction) reaction requires the preparation of DNA denatured by heating at 95°C. This preparation is then annealed with an excess of two complementary oligonucleotides on opposite DNA strands on either side of the sequence to be amplified. Each oligonucleotide then serves as a primer for DNA polymerase (extracted from a thermophilic bacterium such as Thermus aquatis: Tag polymerase) to make a copy of each of the DNA strands. This cycle can be repeated automatically with subsequent denaturations-renaturations.

There are many references describing the PCR protocols in detail: US Patent Nos. 4,683,192, 4,683,202, 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., ed. Academic Press, San Diego, California (1990).

The DNA library is preferably a cDNA library.

More preferably, said oligonucleotide OX can be detected using a labeling agent such as ³² P or digoxigenin.

2

Particularly preferred will be a method for identifying precursors of an amidated polypeptide hormone characterized in that the amplification step uses a combinatorial library of oligonucleotides OX and a further combinatorial library of oligonucleotides 02.

The invention also relates to a method for identifying a peptide precursor having an amidated C-terminal end, which comprises the following steps:

obtaining a DNA bank, using the PCR technique to amplify the fragment of interest using a group of OX oligonucleotides, identifying a DNA sequence, of the given bank, which hybridizes with the OX oligonucleotide and which has been amplified by PCR. reaction, identifying in this sequence one or more peptides with a possible amidated C-terminus.

The aim of this method is to characterize nucleotide sequences that code for precursors containing more than one amidation site.

The DNA bank is preferably a cDNA bank.

More preferably, a given OX oligonucleotide can be detected using a labeling agent such as ³² P or digoxigenin.

Particularly preferred will be a method for identifying precursors of amidated polypeptide hormone characterized by

3

in that the amplification step uses a combinatorial library of OX oligonucleotides.

Another method for identifying a polypeptide precursor containing an amidated C-terminal end, proposed by the present invention, is characterized by the following steps:

obtaining a DNA bank, using the PCR technique to amplify the fragment of interest using the OX oligonucleotide and another single-stranded OX oligonucleotide capable of hybridizing under mild or stringent conditions with a universal consensus sequence contained in the sequence of the plasmid vector in which the DNAs of the given DNA bank are cloned, such as primers T3, T7, KS, SK, M13, Reverse, identifying the DNA sequence of the given bank that hybridizes with the OX oligonucleotide, identifying in this sequence one or more peptides with a possible amidated C-terminal end.

The universal consensus sequence is the sequence carried by the vector into which the DNA library is cloned. This sequence can serve as a primer for sequencing. The nucleotide sequences of these primers are available in J. Sambrook, E. F. Fritch, T. Maniatis, ” Molecular cloning, a laboratory manual, 2nd edition, 1989, Colei Spring Harbor Laboratory Press.

The PCR reaction requires that two 1-nucleotides be

fixed to the cDNA cloned in the vector in order to amplify it. In the case where only a single sequence belonging to the DNA fragment to be amplified is known, a solution to overcome this problem is to use an oligonucleotide that can hybridize to the nucleotide sequence belonging to the vector in which the cDNA was cloned, such as a universal consensus sequence.

The DNA library is preferably a cDNA library.

The 1-nucleotide OY will be preferred, which can be detected using a labeling agent such as ³² P or digoxigenin.

An amplification step using a consensus library of OX oligonucleotides will be particularly preferred.

Examples of embodiments of the invention

The method described in the invention has been declared valid by its application to an already isolated hormone. The neurohormone chosen is cholecystokinin (CCK), which is the neurotransmitter that is quantitatively most abundant in the brain.

1.1. Preparation of DNA template used for PCR reactions from the commercial Lambda Zapp II library (Rat Brain cDNA Library Vector ref. number 936 501) from STRATAGENE (Lafolla, USA).

This Stratagene cDNA library contains cDNA clones from rat brain cells.

1.1.1. Release of cloned cDNA in the form of Bluescript phagemids (Stratagene, Lajolla, USA)

This is done according to the following protocol: 250 μΐ of cDNA bank at a concentration of 2.10® PFU/ml, 200 μΐ XL blue bacteria (genotype: recA1 endA1 gyrA96 thi~1hsdR17 supE44 relA1 lac/F'proAB lacPZ/X M1 5Tn 1 0( Tet ^r ) / ^c ~ compare Bullock, Fernandez, Short, Biotechniques, 5, 376 to 379 (1987) - optical density at 600 nm: OD - 2.5) and 1 μΐ of ExAssist™ tag (see B.Hay, J.Short, Strategies, 5, 16 to 18 (1992)) at a concentration of 10 ¹⁰ PFU/ml are brought into contact for 15 minutes at 37 °C. The entire system is then incubated in 50 ml of LB medium (composition: a mixture of 10 g of sodium chloride, 5 g of yeast extract and 10 g of Bactotrypton per 1 liter of sterile physiological water) for 3 hours at 37 ⁰ C. The culture medium is centrifuged and the supernatant is then activated by heating at 70 °C for 20 minutes.

1.1.2 Obtaining cDNA in the form of a double-stranded plasmid bank

This step requires incubation at 37°C for 15 minutes with 100 μΐ of the activated supernatant and 200 μΐ of SOLR™ bacteria (genotype: e14'(McrA') /\ (mcrCB-hsdSMR-mrr)171 sbsC recB recd uvrC umuC::Tn5 (Kan^lac gyrA96 relA1 thi-1 endA1 lambda^/F' proAB lacPZ/X M15/ ^c Su' (non-inhibitory), see B.Hay, JMShort, Strategies 5(1), 16 to 18 (1992) ~ OD = 1 to 600 nm). After adding 50 μΐ of ampicillin (100 mg/ml) and 50 ml of LB medium, the whole system was incubated overnight at 37°C with constant stirring. Plasmids were prepared from 50 ml of culture using the QIAGEN Plasmid Midi Kit protocol and columns from QIAGEN (QIAGEN columns contain an anionic ion exchanger with positively charged diethylamino~ethanol groups on their surface, which react with the phosphates of the DNA backbone). This resulted in a DNA solution with a concentration of 1.37 pg/μΐ.

6

1.2. Amplification of a part of the CCK precursor from the plasmid bank prepared in this way

1.2.1. Generating the sequences of two oligonucleotides required for the PCR reaction

One of these two nucleotides will contain a sequence complementary to that coding for the CCK amidation site, which is known to have the sequence G1y~Arg-Arg-Ser~A1a~G1u. This oligonucleotide, which will be called oligo CCK amide, has as its nucleotide sequence:

5'CTCAGCACTGCGCCGGCC 3'

The second oligonucleotide, called oligo CCK 5', corresponds to the consensus signal sequence:

5'GTGTGTCTGTGCGT6GTG 3'

The size of the expected amplification product is 315 base pairs, which is the distance between the sequences that corresponds to the two 1 nucleotides on the precursor CCK sequence.

1.2.2. PCR reaction

Prepare a diluted solution D1 containing 1 μΐ of Goldstar Taq polymerase enzyme 5 U/μΙ (see P.Reynier, J.F.Pe11 issier, J.R.Harle, Y.Malthiéry, Biochemical and Biophysical Research Communications, 20.5 (1), 375 to 380 (1994)), 1 μΐ of buffer 10-fold concentrated in standard Taq polymerase and 8 μΐ of water.

Then, 1 μΐ oligo CCK 5' at a concentration of 250 ng/μΐ, 1 μΐ oligo CCK amide at a concentration of 250 ng/μΐ, 1 μΐ dNTP at a concentration of 10 mM each, 1 μΐ cDNA library at a concentration of 250 ng/μΐ, 5 μΐ buffer concentrated 10~fold in Taq polymerase enzyme, 2 μΐ magnesium chloride at a concentration of 25 mM, 1 μΐ diluted D1 solution and 37 μΐ water are mixed.

The amplification conditions are as follows: first, heat treatment is performed for 5 minutes at 95°C, then 30 cycles are repeated. Denaturation is performed at 95°C for 45 seconds, annealing at 60°C for 30 seconds and extension for 1 minute at 72°C. Finally, an additional cycle is performed at 72°C for 10 minutes with a 1-fold extension.

1.2.3. Results

The results are read by migration on a 0.8% agarose gel from 1/10 of the PCR product. reaction. In the presence of 3,8™diamino-5-ethyl-6“-phenylphenanthridinium bromide (ethidium bromide), one intense band is visualized on the gel with a size slightly larger than the labeling substance with a molecular weight of 300.

1.3 Subcloning of the PCR product into a vector allowing sequencing

The vector used is pGEM T-easy Vector (supplied by PROGEMA Corporation, Madison, USA, ref. designation A 1380 ~ sequence given in Appendix I). The steps are as follows:

- purification of the band corresponding to the PCR product by electroelution,

- overnight ligation at 16°C with 1 μΐ of pGEM T-easy vector at 50ng/μl and 1 μΐ of the buffer used in ligation concentrated 10-fold.

- extraction of 3 μΐ of product from the purified band estimated at 20ng/pl, ~ replenishment with water to 10 μΐ.

Bacteria JM 109 (genotype: e14-(McrA) recA1 endA1 gyrA96 thi~1 hsdR17(rk“mk+) supE44 relA1 ,A( 1 ac-pr o AB) /F ' traD36 proAB 1 aď’ Z.Z.\M1 5/-vi z C . Yan i sh-Perron, J.Viera, J.Messing, Gene, 33, 103 to 199 (1985)) were made competent by pretreatment with calcium chloride and then transformed by heat shock for 45 seconds at 45 »C with 1/5 of the ligation mixture. The cells were then cultured overnight in LB-ampicin 1 i nu medium in Petri dishes at 37 °C.

DNA plasmids are prepared by recombining several clones.

Subcloning is then verified by enzymatic digestion with Eco RI.

1.4 Sequence of operations

It is performed using the classical SANGER dideoxynucleotide technique on the pGEM T-easy Vector, incorporating 315 base pairs of PCR product (prepared on a large scale using the QIAGEN tip 100 kit). The primer used for sequencing is the universal T7 oligonucleotide present on the pGEM plasmid.

T-easy Vector.

1.5 Result

The following raw sequences were obtained

GTG TGT CTG TGC GTG GTG ATG GCA GTC CTG GCA GCA GGC GCC CTG GCG CAG CCG GTA GTC CCT GTA GAA GCT GTG GAC CCT 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 GAC TAC ATG GGC TGG ATG GAT TTC GGC CGG CGC AGT GCT GAG

The translation of the sequence obtained in amino acids results in:

VCLCVV MAVLAAGALA QPVVPVEAVD PMEQRAEEAP

RRQLRAVLRP DSEPRARLGA 'LLARYIQQVR KAPSGRMSVL

KNLQGLDPSH RISDRDYMGW MDFGRRSAE which allows easy finding of the nucleotide sequence of the OCR precursor (sequence which was provided by the Swiss databank Swiss prot no. p 0 1 3 5 5).

The abbreviations of amino acids are as follows:

A1 a n i n A Leucine L Arg i n R Lys i n To Aspartic acid D Methionine M Asparagine N Phenylalanine F Cysteine C For 1 in P Acid and glutamic E Serine With

G1utami η Q Threon i n γ G1 yc i η G Tryptophan w Hi st i d i η H Tyros i n Y I am 1euc i η I Valine in

Claims (25)

1 to 7 or oligonucleotides 02 according to claim 14, characterized in that it creates a combinatorial library. 1. A single-stranded oligonucleotide OX, characterized in that it contains 9 to 42 nucleotides and under mild conditions is capable of hybridizing with an oligonucleotide OY of the sequence Y1-Y2-Y3-Y4-Y5, in which Y1 represents a nucleotide sequence of 1 to 12 nucleotides or Y1 is suppressed, Y2 represents a trinucleotide that codes for Gly, Y3 and Y4 independently represent a trinucleotide that codes for Arg or Lys and Y5 represents a nucleotide sequence of 1 to 21 nucleotides or Y5 is suppressed. 2 4 2. Oligonucleotide OX according to claim 1, characterized in that it contains 9 to 42 nucleotides and is capable of hybridizing under stringent conditions with oligonucleotide OY of the sequence Y1-Y2-Y3-Y4-Y5, in which Y1 represents a nucleotide sequence of 1 to 12 nucleotides or Y1 is suppressed, Y2 represents a trinucleotide that codes for Gly, Y3 and Y4 independently represent a trinucleotide that codes for Arg or Lys and Y5 represents a nucleotide sequence of 1 to 21 nucleotides or Y5 is suppressed. 3. The OX oligonucleotide according to claim 1 or 2, characterized in that Y1 is suppressed in the OY oligonucleotide. 4. The OX oligonucleotide of claim 1, 2, or 3, wherein Y5 is suppressed in the OX oligonucleotide. OY. 5. The OX oligonucleotide according to claim 1, 2 or 3, characterized in that Y5 represents a nucleotide in OY. the sequence Y6-Y7-Y8-Y9, in which Y6 represents a trinucleotide that codes for Ser, Thr or Tyr, Y7 represents a trinucleotide that codes for any amino acid, Y8 represents a trinucleotide that codes for Glu or Asp and Y9 represents a nucleotide sequence containing 1 to 12 nucleotides. 6. The OX oligonucleotide according to claim 5, characterized in that Y1 and Y9 are suppressed in the OY oligonucleotide. 7. The OX oligonucleotide according to claim 6, characterized in that it can hybridize with a given OY oligonucleotide, in which Y2 represents a trinucleotide that codes for Gly, Y3 represents a trinucleotide that codes for Lys, Y4 represents a trinucleotide that codes for Arg and Y5 represents a sequence of three trinucleotides that code for Ser A 1 and —G 1 u . 8. a single-stranded oligonucleotide OY, characterized in that it contains 9 to 42 nucleotides of the sequence Y1-Y2-Y3-Y4-Y5, in which Y1 represents a nucleotide sequence of 1 to 12 nucleotides or Y1 is suppressed, Y2 represents a trinucleotide that codes for Gly, Y3 and Y4 independently represent a trinucleotide that codes for Arg or Lys and Y5 represents a nucleotide sequence of 1 to 21 nucleotides or Y5 is suppressed. 9. The OY oligonucleotide of claim 8, wherein Y1 is suppressed. 10. Oligonucleotide OY according to claim 8 or 9, characterized in that Y5 is suppressed. 11. The oligonucleotide OY according to claim 8 or 9, characterized in that Y5 represents the nucleotide sequence Y6-Y7-Y8-Y9, in which Y6 represents a trinucleotide that codes for Ser, Thr or Tyr, Y7 represents a trinucleotide that codes for any amino acid, Y8 represents a trinucleotide that codes for Glu or Asp and Y9 represents a nucleotide sequence containing 1 to 12 nucleotides. 12. The OY oligonucleotide according to claim 11, wherein Y1 and Y9 are suppressed. 13. The OY oligonucleotide according to claim 12, wherein Y2 represents a trinucleotide encoding Gly, Y3 represents a trinucleotide encoding Lys, Y4 represents a trinucleotide encoding Arg and Y5 represents a sequence of 3 trinucleotides encoding Ser-Ala-Glu. 14. A single-stranded oligonucleotide O2, characterized in that it contains 15 to 39 nucleotides and is capable of hybridizing under mild or stringent conditions with a consensus signal sequence characteristic of amidated polypeptide hormones, said sequence having the formula 21-22-23-24-25-26-27, in which 21 represents a nucleotide sequence of 1 to 12 nucleotides or 21 is suppressed, 22 and 23 represent two trinucleotides that code for Leu, 24 and 25 represent two trinucleotides that code for any two amino acids, 26 represents a trinucleotide that codes for Leu and 27 represents a nucleotide sequence of 1 to 12 nucleotides or 27 is suppressed. 15. A group of 1 OX oligonucleotides according to any one of claims 16. A method for identifying a peptide precursor having an amidated C-terminal end, characterized in that it comprises the following sequential steps: ~ obtaining a DNA bank, - hybridizing one or more oligonucleotides according to any one of claims 1 to 7 with a given DNA library, - identifying the DNA sequence or sequences of the library that hybridizes with the oligonucleotide according to any one of claims 1 to 7 and - identification in this sequence or sequences of one or more peptide precursors with a possible amidated C-terminal end. 17. The method of claim 16, wherein the hybridization step uses a combinatorial library of claim 15. 18. A method for identifying a peptide precursor having an amidated C-terminal end, characterized in that it comprises the following sequential steps: - obtaining a DNA bank, - using the PCR technique to amplify the fragment of interest using a group of oligonucleotides according to any one of claims 1 to 7 and another group of oligonucleotides according to claim 14, - identification of a DNA sequence or sequences of a given library which hybridizes with an oligonucleotide according to any one of claims 1 to 7 and - identification in this sequence or sequences of one or more peptide precursors with a possible amidated C-terminal end. 19. The method of claim 18, wherein the amplification step uses the combinatorial libraries of claim 15. 20. A method for identifying a peptide precursor having an amidated C-terminal end, comprising the following steps: - obtaining a DNA bank, - using the PCR technique to amplify the fragment of interest using a group of 1 oligonucleotides according to any one of claims 1 to 7, - identification of a DNA sequence or sequences of a given library which hybridizes with an oligonucleotide according to any one of claims 1 to 7 and ~ identification in said sequence or sequences of one or more peptide precursors with a possible amidated C-terminal end. 21. The method according to claim 20, characterized in that in that the amplification stage uses a combinatorial library according to claim 15. 22. A method for identifying a polypeptide precursor having an amidated C-terminal end, comprising the following steps: ™ obtaining a DNA bank, - using the PCR technique to amplify the fragment of interest using the oligonucleotide according to any one of claims 1 to 7 and another single-stranded oligonucleotide capable, under mild or stringent conditions, of hybridizing with a universal consensus sequence contained in the sequence of the plasmid vector in which the cDNA of the DNA bank is cloned, such as primers T3, T7, KS, SK, M13 and Reverse, - identification of a DNA sequence of a given library which hybridizes with an oligonucleotide according to any one of claims 1 to 7 and ~ identification in this sequence of one or more peptide precursors with a possible amidated C-terminal end. 23. The method of claim 22, wherein the amplification step uses a combinatorial library according to claim 15. 24. The method according to any one of claims 16 to 23, wherein the DNA library is a cDNA library. 25. The method of any one of claims 16 to 24, wherein the single-stranded oligonucleotide can be detected using a labeling agent such as ³² P or digoxigenin.