FR2790004A1 - Use of new and known morpholine nucleotide analogs for labeling nucleic acid fragments, especially for nucleic acid sequencing - Google Patents

Abstract

Morpholine nucleotide analogs (I) are used for labeling DNA or RNA fragments. Morpholine nucleotide analogs of formula (I) are used for labeling DNA or RNA fragments. R<1> = a nucleobase; R<2> = (CH2)nQ; Q = NH2, SH, COOH, OH, NHR<3>, SR<3>, COR<3> or OR<3>; R<3> = a group derived from a label, protein, enzyme, fatty acid or peptide. Independent claims are also included for: (1) the preparation of a 3'-labeled nucleic acid (DNA or RNA) fragment, comprising enzymatic incorporation of a nucleotide derived from (I) at the 3'-terminal hydroxy group of a nucleic acid fragment; (2) a process for modifying a nucleic acid fragment by enzymatic 3'-incorporation of a modified morpholine nucleotide derived from a compound of formula (I) where Q is NHR<3>, SR<3>, COR<3> or OR<3> and R<3> = a photo crosslinker, fatty acid, hydrophobic peptide, antibody, enzyme or fluorophore; (3) a process for sequencing a nucleic acid fragment by synthesizing the complementary strand by enzymatic polymerization using a chain terminator derived from (I); (4) a morpholine nucleotide of formula (I) where R<1> is adenine and R<2> is CH2COOH, (CH2)4NH2 or (CH2)4NH-fluorescein; (5) a morpholine nucleotide of formula (I) where R<1> is thymine and R<2> is CH2COOH; and (6) the preparation of (I).

Description

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MORPHOLINO-NUCLEOTIDES MANUFACTURING PROCESS, AND
USE OF THESE FOR ANALYSIS AND MARKING OF
NUCLEIC ACID SEQUENCES
DESCRIPTION
Technical area
The present invention relates to the manufacture of nucleic acid fragments (DNA or RNA) elongated enzymatically by means of morpholinonucleoside triphosphates. This extension can be used for the analysis of nucleic acid sequences by incorporation of these derivatives into nucleic acid chains as well as enzymatic labeling and immobilization or detection of sequences.

These morpholino-nucleoside triphosphates can still be used with an additional molecule which can play various roles in many applications.

State of the prior art
The most widely used method for analyzing nucleic acid sequences is the so-called chain termination enzymatic technique, developed by Sanger et al in Proceedings of National Academy of Science, 74, 1977, p. 5463-5467 [1]. It is based on the properties that DNA-dependent DNA polymerases have of creating DNA polymers complementary to the sequence of a DNA strand serving as a template, from a mixture of natural nucleoside triphosphates monomers. The

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method consists, from the DNA strand to be analyzed, in making a series of copies of the complementary strand by adding molecules called chain terminators to the conventional reaction medium, then analyzing the length of the newly formed strands to determine the sequence of the bases of the matrix. The principle of the method is explained in Table 1 below.

Table 1

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This Table 1 illustrates what occurs when we bring together the DNA polymerase, a primer consisting of an oligonucleotide of reduced size, generally less than 25 bases, and the mixture of the four natural nucleoside triphosphates with the DNA strand whose sequence we want to determine, which constitutes the matrix. The primer corresponds to the start of the sequence complementary to the DNA strand to be analyzed. From this primer, which spontaneously interacts with the complementary sequence of the DNA strand to be analyzed (hybridization), the enzyme incorporates complementary nucleotides of the matrix to construct by elongation-polymerization a new DNA strand, copy complementary to the latter. The new nucleotides are incorporated exclusively from the 3'-OH terminal end of the growing chain, sequentially and respecting the rules of complementarity between bases of Watson & Crick. A thymine is incorporated into the newly formed strand by complementarity with an adenine present in the strand serving as the matrix, a guanine is incorporated in complementarity with a cytosine and vice versa. If all the necessary compounds are provided in an unlimited amount, the enzyme catalyzes the polymerization of the strand formed until the latter represents all of the perfect complementary strand of the matrix.

On the other hand, if we add to the reaction medium a molecule which is recognized by the polymerase but which does not have a free 3'-OH terminal end, each time this molecule is incorporated, the work of polymerization of the enzyme will be interrupted because the channel will no longer be able to grow due to the absence of a site available to anchor a

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new nucleotide (creation of newly formed interrupted strands). This is illustrated in Table 2 below with 3 'deoxythymidine, 5'-triphosphate.

Table 2

By using this thymidine derivative, which will be called the T chain terminator at an adequate concentration, a series of DNA strands is obtained for a given matrix, the size of which is fixed statistically by the position of the adenines in the matrix. The result

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obtained is illustrated in Table 3. The sequence of the matrix is written in the first line, the sequence of newly formed strands created with the chain terminator T (denoted S) is written in the following lines.

5'- T A C G T A A G G C T G G A G A C T A G S
Dans cet exemple, la matrice comporte
5 adénines dans la zone qui est détaillée, l'ADN polymérase pourra donc produire 5 brins néoformés interrompus, de longueurs différentes. Table 3 MATRIX 3'-ATGCATTCCGACCTCTGATCAG-5 'COPIES OF MATRIX 5'- S 5'- TACGS 5'-TACGTAAGGCS 5'-TACGTAAGGCTGGAGACS

5'- TACGTAAGGCTGGAGACTAGS
In this example, the matrix has
5 adenines in the area which is detailed, the DNA polymerase will therefore be able to produce 5 new interrupted strands of different lengths.

It is then sufficient to analyze this mixture by electrophoresis on polyacrylamide gel in a denaturing medium to determine the length of each of the strands obtained using the T chain terminator. The size of the newly formed interrupted strands makes it possible to deduce the position of the adenines on the matrix. .

By repeating this experiment three times with respectively terminator products of chains A, G and C, a total of four series of DNA fragments are obtained, the length of which makes it possible to determine the entire sequence of the template strand.

The RNA sequencing technology is based on the same principles, the difference being that the enzyme used is a reverse transcriptase (or DNA polymerase-RNA dependent).

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dans laquelle B représente l'une des bases nucléiques A, C, G ou T, comme il est décrit dans le document [1]. The products most used as chain terminators to stop the action of DNA polymerases are 2 ', 3'-dideoxynucleoside triphosphates of the formula:

in which B represents one of the nucleic bases A, C, G or T, as described in document [1].

The structure of these products compared to that of natural nucleoside triphosphates shows the absence of the hydroxyl function in position 3 'which serves as the position of attachment of the next nucleotide.

The chemical synthesis of 2 ', 3'-dideoxynucleotides is carried out according to a long and tedious protocol comprising three main steps. In the case of guanine, the first step in this process is the protection of the exocyclic amine function of guanine and of the primary 5 'hydroxyl function of the sugar. The 3 ′ hydroxyl function is then removed by elimination and then by reduction of the 2′-3 ′ double bond generated. The last step is the preparation of the triphosphate derivative.

Other chain terminators have been described in WO-A-96/23807 [2]. These are the 5'-triphosphates of arabinonucleosides, 3'-fluoro- 2 ', 3'-dideoxynucleosides, 3'-azido-2', 3'dideoxynucleosides or 3'-amino-2 ', 3'-dideoxynucleosides . Their synthesis is just as laborious.

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At the origin of the Sanger method, the visualization of the DNA fragments synthesized was done by radioactive labeling with 32P in 5 'of the primer used to initiate the polymerization of the complementary strand.

A modification was made using primers carrying a fluorophore. This improvement relates only to ease of use, since it eliminates the use of radioactive materials, but four sequencing reactions must still be carried out, each using a different polymerization terminator (terminator A, G, T or C).

A new step has been taken with the use of terminators of sequences carrying fluorophores on their nucleic base, as described by Prober et al, in Science, 238, 1987, pages 336-341 [3].

Under these conditions, the labeling of the neo-synthesized strands is no longer done before the sequencing reaction, but directly at the time of incorporation of the sequence terminator. By taking care to choose a fluorophore with different optical properties for each DNA base, the experimental protocol has been greatly simplified. Only one reaction is carried out with the four terminators mixed. Therefore, from a single electrophoresis channel, the four nucleotides of the sequence are distinguished by virtue of the different emission wavelengths of the four terminators.

This simplification in the analysis protocol does not only have advantages. In fact, the fluorophores are grafted directly onto the base. This structural modification, located in the direct vicinity of the sites of hydrogen bonds governing the recognition between the bases, causes a decrease

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recognition by enzymes. To compensate for this, an increase in the concentration of terminators is recommended, which leads to a very high consumption of the raw material having a very high added value. In addition, the synthesis of these molecules is still as delicate.

Disclosure of the invention
A subject of the present invention is in particular the use, in a sequencing process of this type, of chain terminators consisting of nucleoside triphosphate analogs which are easier to synthesize, which also make it possible to carry out efficient labeling without modifying the nucleic bases. .

dans laquelle R1 représente une base nucléique et R2 représente un groupe répondant à l'une des formules Also, the subject of the invention is a method for sequencing a nucleic acid (DNA or RNA) by the enzymatic polymerization technique of the complementary sequence of this nucleic acid using chain terminators, in which at least one chain terminators has as a precursor a compound corresponding to the formula:

in which R1 represents a nucleic base and R2 represents a group corresponding to one of the formulas

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following: - (CH2) n-NH2 - (CH2) n-SH - (CH2) n-COOH - (CH2) n-OH - (CH2) n-NH-R3 - (CH2) n-SR - (CH2) n-CO-R3 - (CH2) n-OR3 in which n is an integer ranging from 1 to 12 and Rest a group derived from a label, a protein, an enzyme, a fatty acid or of a peptide.

The chain terminators used in this process are nucleotide derivatives comprising a nucleic base R which allows recognition by polymerases and transcriptases, and compliance with the rules of complementarities of Watson and Crick.

The nucleic bases used for R can be natural or synthetic. The natural bases are generally chosen from adenine, guanine, cytosine, thymine, uracil, xanthine, hypoxanthine, 2-aminopurine and their derivatives.

Synthetic bases are analogues or derivatives of natural nucleic bases, which are capable of interacting with natural bases.

Preferably, R corresponds to one of the following formulas:

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In the nucleotide derivatives of formula (I), the osidic part is replaced by a suitably substituted morpholine comprising:
1) A hydroxymethyl function close to cyclic oxygen, esterified with a triphosphoric acid group. This part of the molecule mimics the 4 ', 5' part of the nucleotides and allows the attachment by the polymerase or the transcriptase to the growing DNA or RNA chain.

2) An amine function substituted by R2, which can optionally allow the grafting of a chromophore or of a biologically active group and above all, which prevents the attachment of another nucleotide (interruption of the polymerization).

Compared to the derivatives conventionally used in the Sanger method such as those described in documents [1], [2] and [3], these compounds can be synthesized in a single step directly from ribonucleoside triphosphates, as will be seen. hereafter.

The advantage of these compounds lies in the very large choice of R2 groups (substituents of the morpholine cycle) which can be used which make it possible to functionalize this cycle. Functions such as acids, amines, thiols or ethers can be added and will allow the grafting of various chemical compounds, in particular of markers useful for the identification of DNA or RNA fragments.

The markers used for R3 can be chosen from a very large set of molecules known for the labeling of nucleotides. They can be chosen for example from radioactive products, luminescent, electroluminescent and fluorescent products,

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molecules capable of coupling with other molecules, molecules allowing interactions of the antigen-antibody type, and enzymatic markers.

Preferably, for the sequencing of nucleic acids, R is a fluorophore, for example, chosen from all the derivatives of fluorescein or of rhodamine. One can also use those of biotin. In particular, those of the derivatives used for labeling nucleic acids will be chosen.

Nucleoside derivatives in which the osidic part of the nucleoside has been replaced by a morpholine, have already been synthesized in the prior art, as appears in the following documents: - Hileman et al, Bioconjugate Chemistry, 5,1994, pages 436 -444 [4], - Broker et al, Nucleic Acids Research, 5,1978, pages 363-385 (5], - Agrawal et al, Nucleic Acids Research, 14,1986, pages 6227-6245 [6], - FR -A- 2 710 068 [7], and - Rayford et al, Journal of Biological Chemistry,
260,1985, pages 15708-15713, [8].

The nucleoside derivatives of document [4] contain a morpholino ring which is substituted by a fluorescein or a rhodamine. They are used for the study of proteins and not as chain terminators in a nucleic acid sequencing process.

Their manufacture differs from the process we report, as the fluorophore is incorporated directly on the morpholine ring. The technique that we describe uses an intermediate purification step which

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allowed us to isolate and perfectly characterize the final product, unlike Hileman et al.

In document [5], it is transfer RNA modified at its 3 'end by a nucleoside derivative comprising a morpholine ring substituted by a biotin. This product is used as a chemical marker for transfer RNAs to study the chromosomal localization of transfer RNA genes.

In document [6], it is an oligonucleotide comprising a morpholine ring coupled to a biotin, which is used as a probe for the detection and isolation of specific genes.

Document [7] describes nucleoside derivatives comprising a substituted morpholine ring. They are used for the preparation of antibodies directed against a hapten attached to the morpholine ring of the nucleoside derivative.

Document [8] illustrates a morpholinoadenosine substituted with CH2COOH, used for affinity chromatography.

Thus, none of these documents relates to the use of nucleotide derivatives such as those of the invention, as chain terminators, in a method for sequencing nucleic acids according to the Sanger method.

The nucleotide derivatives used in the process of the invention can be prepared in a single step, directly from the ribonucleoside triphosphates according to the following reaction scheme illustrated with R representing adenine.

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This process is of the same type as the processes described in documents [6] and [7] for forming the morpholino ring.

It is also possible to prepare the nucleotide derivatives of formula (I) from morpholinonucleosides and then introduce the triphosphate group using the Eckstein protocol, as described by Ludgwig et al in J. Org. Chem. 54, 1989, pages 631-635 [9].

The enzymes which can be used for the enzymatic polymerization can be those described below.

dans laquelle Ra la signification donnée ci-dessus, avec un periodate, un composé de formule R2 NH2 dans laquelle R2 a la signification donnée ci-dessus et du borohydrure de sodium. According to the invention, the process for preparing morpholino-nucleotides of formula (I) comprises the reaction of a nucleoside triphosphate of formula:

in which Ra has the meaning given above, with a periodate, a compound of formula R2 NH2 in which R2 has the meaning given above and sodium borohydride.

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The invention also relates to the use of a nucleotide derivative having as a precursor a compound of formula (I) for the 3 'labeling of nucleic acid fragments (DNA or RNA) by enzymatic incorporation of the nucleotide derivative into it. 3'OH end of the nucleic acid fragment.

It also relates to the process for the frabrication of a 3 ′ labeled nucleic acid fragment (DNA or RNA) by enzymatic incorporation of the nucleotide derivative mentioned above at the 3′OH end of the nucleic acid fragment.

The enzyme used can be the Klenow fragment of DNA polymerase, and a template or template is then used to attach the morpholino-nucleoside to the nucleic acid fragment which serves as a primer.

The enzyme used can also be a heat-resistant polymerase of a thermophilic bacterium or the terminal transferase or the reverse transcriptase.

The DNA or RNA fragments thus labeled can be used to block any subsequent ligation and provide protection against exonucleases, as well as for detecting DNA or RNA fragments.

It is also possible to use a modified morpholino-nucleotide having as a precursor a compound of formula (I) to modify a fragment of nucleic acid (DNA or RNA) by enzymatic incorporation at the 3 'end thereof of a morpholino- modified nucleotide having as precursor a compound of formula (I) comprising in R3 a compound chosen from photocrosslinking agents, for example for crosslinking on DNA or on any support; fatty acids, hydrophobic peptides, antibodies, for example to facilitate penetration into cells, enzymes or parts of enzymes such as alkaline phosphatases, peroxidases, acetylcholinesterases for detection, restriction enzymes for cleavage of vicinal DNA, and fluorophores.

As previously, the incorporation of this modified morpholino-nucleotide is carried out enzymatically. The nitrogenous bases, the markers and the enzymes which can be used can be the same as those mentioned above.

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According to the invention, the nucleotide derivative, the modified morpholino-nucleotide and the chain terminator used respectively for the 3 'labeling of nucleic acid fragments, for the modification of nucleic acid fragments or for the sequencing of nucleic acid fragments. a nucleic acid, can be the compound (I) in the form of monophosphate.

Other characteristics and advantages of the invention will become more apparent on reading the following description of exemplary embodiments, of course given by way of illustration and not limitation, with reference to the appended drawing.

Brief description of the figures
FIG. 1 is a diagram illustrating the results obtained for the sequencing of plasmid DNA with the chain terminator of the invention (solid line curve) and with the chain terminator of the prior art (dashed line).

FIG. 2 is a diagram illustrating the results obtained by testing morpholino A putrescine (MATPP) and morpholino A fluorescein (MATPPF) in sequencing.

FIG. 3 is a diagram illustrating the result on polyacrylamide gel of a test making it possible to follow the elongation of an oligonucleotide A and the incorporation of morpholino A putrescine.

Detailed description of the embodiments
Examples 1 to 4 which follow illustrate the synthesis of morpholino-nucleotides of formula (I).

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Example 1: Synthesis of 4- (carboxymethyl) -2- (adenosin-9-yl) -6- (hydroxymethyl) morpholine-6-triphosphate (morpholino A glycine).

This morpholino A glycine corresponds to formula (I) in which R1 is adenine and R2 is the group -CH2-COOH.

In this example, all the reactions are carried out at room temperature, with magnetic stirring, in a 50 mL flask.

1.000 g (1.8 mmol, 1 eq.) Of 5 ′ adenosine triphosphate are dissolved in 10 ml of water, then 1 eq. sodium periodate (388 mg, 1.8 mmol). The solution is then stirred for 35 minutes.

Glycine (682 mg, 9.1 mmol, 5 eq.) In solution in 2 mL of water (pH = 9.5-10) is added and the pH of the solution is raised to 9.5-10 with solid potassium carbonate. The solution is stirred for 55 minutes. The reaction mixture turns yellow.

Sodium borohydride (total 166 mg, 4.4 mmol, 2.5 eq.) Is added in six equivalent fractions, each dissolved in 0.2 mL of water. After adding the first, gas evolution is noted. The other fractions, each dissolved just before addition, are added every hour.

After one night, the solution is neutralized by adding 1M formic acid to pH 4-5, then it is evaporated.

Reverse phase chromatography analysis on a Merck LiChrocart 125-4 LiChrospher 100 RP-18 column ("endcapped", 5 m, 125x4 mm) using a flow rate of 1 mL / min and the eluent acetate.

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25mM TEAA triethylammonium / MeOH methanol [98/2], indicates a yield of 40% (k '= 3.85).

Purification: it is done by preparative high performance liquid chromatography (HPLC) using a Macherey Nagel Nucleosil 7 C-18 column
7 μm, 250x21 mm) with a flow rate of 8 mL / min and 25 mM TEAB triethylammonium bicarbonate, as eluent.

Characterization: - 1H NMR: 8 (ppm): 8.47 (s, 1H, H2), 8.37 (s,
1H, H8), 6.26 (dd, 1H, H1 '), 4.54 (m, 1H, H4'), 4.28 (m,
1H, H5 "), 4.22 (m, 1H, H5 '), 3.70 (m, 1H, H2'), 3.68 (s,
2H, CH2-Glycine), 3.41 (m, 1H, H2 "), 3.45 (m, 1H, H3 '),
3.30 (m, 1H, H3 "), - 13C NMR. # (Ppm): 152.7 (C2), 140.5 (C8), 78.6 (Cl '), 74.1 (C4') , 66.4 (C5 '), 60.6 (CH2), 54.5 (C2'), 53, 6 (C3 ') - 31P NMR. 5 (ppm): -6.44 (d, 1P, yP ), -11, 68 (d, 1P, aP), -22, 11 (t, 1P, PP) - Mass spectrometry: MH = 547.04 g.mol-1
The other three morpholino-nucleotides with R1 representing thymine, guanine and cytosine can be synthesized in an analogous manner.

Example 2: Synthesis of 4- (aminobutyl) -2- (adenosin-9yl) -6- (hydroxymethyl) morpholine-6-triphosphate (morpholino A putrescine).

This putrescine morpholino A corresponds to formula (I) with R1 representing adenine and R representing the group - (CH2) 4-NH2.

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All the reactions are carried out at room temperature, with magnetic stirring, in a 100 mL flask.

5'-adenosine triphosphate (500 mg, 0.9 mmol, 1 eq.) Is dissolved in 10 mL of water, then 1 eq is added. sodium periodate (194 mg, 0.9 mmol). The solution is then stirred for 45 minutes.

Putrescine (456 µL, 4.5 mmol, 5 eq.) Is added. The solution is stirred for 45 minutes. The reaction mixture turns yellow.

One sixth of sodium borohydride (in total 86 mg, 2.3 mmol, 2.5 eq.) Dissolved in 0.1 ml of water is added to the solution. Gas evolution is noted. The other sixths, each dissolved just before addition, are added every hour.

After one night, the solution is neutralized by adding 1M formic acid to pH4-5, then it is evaporated.

An analysis is carried out by reverse polarity chromatography on a Merck LiChrocart 125-4 LiChrospher 100 RP-18 column ("endcapped", 5 μm, 125x4mm) with a flow rate of 1 mL / min, using a 25mM TEAB gradient as eluent. / MeOH, under the following conditions: t (min) TEAB (%) MeOH (%)
0 97 3
2 97 3
10 90 10
15 90 10
17 97 3

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This analysis indicates a yield of 67% (k '= 3.81).

The product is purified by semi-preparative HPLC on the Phenomenex Ultremex 5-C18 column (250x10 mm) with a flow rate of 4 mL / min, and using as eluent a 25 mM TEAB / MeOH gradient, under the following conditions: t ( min) TEAB (%) MeOH (%)
0 95 5
3 95 5
8 90 10
10 95 5 Characterization - 1H NMR: 8 (ppm): 8.44 (s, 1H, H2), 8.33 (s, 1H, H8), 6.06 (dd, 1H, Hl '), 4.35 (m, 1H, H4 '), 4.22 (m, 2H, H5', H5 "), 3.39 (d, 1H, H2 '), 3.22 (t, 1H, H3"), 3, 14 (s, 2H, CH2 putrescine), 2.92 (t, 1H, H2 '), 2.74 (s, 2H, CH2 putrescine), 2.54 (t, 1H, H3'), 1.78 ( s, 4H, (CH2) 2 putrescine).

- 31 P NMR: 8 (ppm) -8.45 (dd, 1P, yP), -13.25 (dd, 1P, aP), -24.20 (t, 1P, ssP) - Mass spectrometry: MH + = 561.92 g.mol-1

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Example 3 Synthesis of 4- [5 2-aminobutyl) - thioureidyl) fluorescein)] - 2- (adenosin-9-yl) -6- (hydroxymethyl) -morpholine-6-triphosphate (morpholino A putrescine-fluorescein).

This compound corresponds to formula (I) with R representing adenine, and R2 representing (CH2) 4NHR where R is a group derived from fluorescein.

All the reactions are carried out at room temperature, with magnetic stirring, in a 100 mL flask.

Is added three times and gradually to 200 mg (0.3 mmol, 1 eq.) Of Morpholino A putrescine of Example 2, in a water / pyridine mixture (1/1), 184.9 mg (0, 5 mmol, 1.5 eq.) Of fluorescein isothiocyanate. The medium is stirred for 48 hours before being evaporated to dryness.

Analysis by reverse-phase chromatography on a Merck LiChrocart 125-4 LiChrospher 100 RP-18 column ("endcapped", 5 µm, 125x4 mm), with a flow rate of 1 mL / min using as eluent: TEAA 25mM / MeOH [97/3], indicates a yield of about 48% (k '= 7.51).

Purification: it is carried out by “flash” chromatography on a column of silica with reversed phase polarity C-18 (Econosil prep 90, Alltech, France). The eluent is a water / MeOH gradient.

Characterization: - 1H NMR: # (ppm): 8.57 (s, 1H, H2), 8.31 (s, 1H, H8), 8.20-6.65 (9H, fluorescein), 5.79 ( dd, 1H, Hl '), 4.25 (m, 1H, H4'), 4.11 (m, 2H, H5 ', H5 "), 3.60 (s,

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2H, CH2 putrescine), 3.12 (d, 1H, H3 "), 2.93 (d, 1H, H2"),
2.81 (m, 1H, H2 '), 2.59 (m, 2H, CH2 putrescine), 2.50 (dd,
1H, H3 '), 1.79 (s, 2H, CH2 putrescine), 1.62 (m, 2H, CH2 putrescine) - 31P NMR: 5 (ppm): -8.45 (dd, 1P, yP), -13.25 (dd, 1P, aP), -24.20 (t, 1P, ssP)
Mass spectrometry: MH = 949.2 g.mol-1 Example 4: Synthesis of 4- (carboxymethyl) -2- (thymidin-
9-yl) -6- (hydroxymethyl) morpholine-6-triphosphate (morpholinoT glycine).

This compound corresponds to formula (I) with R1 representing thymine and R representing the group -CH2-COOH.

In this example, the morpholino-nucleoside is first prepared, then it is converted into triphosphate. a) Preparation of the morpholino-nucleoside of ribothymidine.

All the reactions are carried out at room temperature, with magnetic stirring, in a 250 mL flask.

Ribothymidine (3.500 g, 13.5 mmol, 1 eq) is dissolved in 35 mL of water, then 1 eq is added. sodium periodate (2.900 g, 13.5 mmol). The solution is then stirred for 45 minutes.

Glycine (5.089 g, 67.8 mmol, 5 eq) in 35 mL of water (pH = 9.5-10) is added and the pH of the solution is raised to 9.5-10 with carbonate of

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potassium. The solution is stirred for one hour and
45 minutes. The reaction mixture turns yellow.

One sixth of sodium borohydride (in total
1.280 g, 33.8 mmol, 2.5 eq) dissolved in 3.5 mL of water is added to the solution. Gas evolution is noted. The other sixths, each dissolved just before addition, are added every hour.

After one night, the solution is neutralized by adding 1M formic acid to pH4-5, then it is evaporated.

An analysis by reverse polarity chromatography on a Merck LiChrocart 125-4 LiChrospher 100 RP-18 column ("endcapped", 5pm, 125x4 mm), with a flow rate of 1 mL / min, using as eluent: TEAA 25mM / MeOH [99/1], indicates a yield of 32% (k '= 8.83).

Purification: it is carried out by “flash” chromatography on a column of silica with reversed phase polarity C-18 (Matrex, Amicon). The eluent is water.

Characterization: - 1H NMR: 8 (ppm): 7.77 (s, 1H, H6), 5.92 (dd, 1H, Hl '), 4.07 (m, 1H, H4'), 3.77 ( m, 2H, H5 ', H5 "), 3.22 (s, 2H, CH2 glycine), 3.13 (dd, 1H, H2"), 2.99 (dd, 1H, H3 "), 2.51 (t, 1H, H2 '), 2.34 (t, 1H, H3'), 1.98 (s, 3H, CH3 base) b) Preparation of the morpholino-nucleoside monophosphate of ribothymidine
To 342 mg of imidazole (5.0 mmol, 3 eq) dried in a desiccator and then taken up in 5 mL of strictly anhydrous pyridine, are added 234 L

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phosphorus oxychloride trichloride (2.5 mmol,
1.5 eq.). The mixture is placed under stirring for
30 minutes in dry air.

At the same time, 500 mg of morpholinothymidine (1.7 mmol, 1 eq.) Obtained in a) are dried 3 times in pyridine, then taken up in 5 ml of anhydrous pyridine.

The imidazole / POC13 / pyridine mixture under argon is added to the morpholinonucleoside solution and the whole is stirred for 48 hours at room temperature. Then 100 µl of water are added, taking care to cool the reaction flask in an ice bath. The reaction mixture is evaporated to dryness then taken up twice with water and evaporated in order to remove the pyridine.

Analysis by reversed-phase chromatography on a Macherey Nagel Nucleosil 5 C-18 column (7 μm, 120 × 3 mm), at a flow rate of: 1 mL / min, using as eluent: TEAA 25 mM-MeOH [97/3] , indicates a yield of 33% (k '= 0.62).

Purification: it is carried out by preparative HPLC on H: Macherey Nagel Nucleosil 7 C-18 column (7 m, 250 × 21 mm) at a flow rate of 5 mL / min using water as eluent.

Characterization: - 1H NMR: 8 (ppm): 7.80 (s, 1H, H6), 5.95 (dd, 1H, Hl '), 4.19 (m, 1H, H4'), 3.94 ( t, 2H, H5 ', H5 "), 3.28 (s, 2H, CH2 gl ycin), 3.24 (m, 1H, H2"), 3.10 (m, 1H, H3 "), 2, 53 (t, 1H, H2 '), 2.39 (t, 1H, H3'), 2.00 (s, 3H, CH3 base)

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- 31P NMR: # (ppm): 1.74 (s) c) Preparation of ribothymidine morpholino-nucleoside triphosphate.

1.097 g of carbonyldiimidazole (6.7 mmol, 5 eq.) Dissolved in 5 ml of anhydrous dimethylformamide are added to the tributylammonium salt of the morpholinonucleoside monophosphate of thymine obtained in b) (511 mg, 1.3 mmol, 1 eq.) dissolved in 3 mL of anhydrous dimethylformamide. The mixture is placed under stirring at room temperature for five hours. The excess of carbonyldiimidazole is destroyed by adding 436 L of methanol (10.8 mmol, 8 eq.). After 30 minutes, 5 equivalents of tributylammonium pyrophosphate (3.008 g, 6.7 mmol) in solution in 5 mL of dimethylformamide are added. The mixture is placed under stirring for two days, then the reaction mixture is filtered and evaporated to dryness.

Reverse phase chromatography analysis is performed on an SFCC PVDI 31 column (5 µm, 100x4.6mm), at a flow rate of 1 mL / min, using an ammonium formate (FA) gradient as eluent. , under the following conditions: t (min) FA 25 mM FA 0.9 M (%) (%)
0 100 0
10 100 0
40 0 100
41 0 100
43 100 0
This indicates a yield of 27% (k '= 13.84).

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Purification: it is carried out by "flash" chromatography on an ion exchange phase column (DEAE
Sepharose Fast Flow, Pharmacia Biotech). The eluent is a TEAB gradient (from 25 mM to 0.9 M).

Characterization: - 1H NMR: # (ppm): 7.74 (s, 1H, H6), 5.92 (dd,
1H, H1 '), 4.25 (m, 1H, H4'), 4.15 (m, 2H, H5 ', H5 "), 3.81 (s, 2H, CH2 glycine), 3.54 (d , 1H, H2 "), 3.10 (t, 1H, H3"), 2.56 (t, 1H, H2 '), 2.45 (t, 1H, H3'), 1.95 (s, 3H , CH3 base) - 31P NMR. # (Ppm): -10.03 (d, 1P, yP), -10.88 (d, 1P, aP), -22.65 (t, 1P, PP) - Spectrometry mass: MH + - 554.48 g.mol-1 Example 5: Use of morpholino T glycine for the analysis of a DNA sequence.

The morpholino T glycine of Example 4 is tested in sequence reaction with fluorescent primers (Applied Biosystems, Perkin-Elmer, Foster City, CA, USA) on a standard template which is Bluescript plasmid DNA (Stratagene, La Jolla, CA, USA). The enzyme used is a Taq Polymerase (Perkin-Elmer) which is used in its buffer (TACS buffer, Perkin-Elmer).

Two reactions were carried out with morpholino T glycine at 200 and 500 M (Table 4), as well as two control reactions (Table 5) with dideoxynucleotide T (Boehringer).

The reaction medium with a total volume of 10 µL contains 125 ng of template, 1.25 pmoles of primer

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fluorescent and the other constituents given in Tables 4 and 5. The medium is subjected to thermal cycles in order to produce a number of molecules of newly formed DNA strands. Amplification on a Perkin-Elmer 9700 apparatus is carried out, according to the following sequences: 3 min., 95 C; 15 cycles (15 sec., 95 C; 15 sec., 55 C; 1 min., 70 C); 15 cycles (15 sec., 95 C; 1 min., 70 C). The amplification product is purified on a Sephadex G50 column.

Migration of the amplification product obtained in the column eluate is carried out in a denaturing gel (7M urea) of acrylamide of the Long Ranger type (6%), in 1X TBE, on an Applied Biosystems 377 apparatus. The electrophoresis is carried out. takes place for 12 hours under 1500 V.

The preparation of the stock solution of nucleotides representing here a mixture of the four natural nucleoside triphosphates, depleted in thymidine triphosphate (called dTTP mix) is carried out as follows.

2 L of a 1.25 mM solution of dTTP (Promega) are mixed with 2 L of 5 mM dATP (Promega), 2 L of 5 mM dCTP (Promega) and 2 L of 5 mM dGTP (Promega).

Table 4

<tb>
<tb> Morpholino <SEP> T <SEP> glycine <SEP> 200 <SEP> M <SEP> Morpholino <SEP> T <SEP> glycine <SEP> 500 <SEP> pM
<tb> Buffer <SEP> TACS <SEP> (x5) <SEP> 2 <SEP> L <SEP> 2 <SEP> L
<tb> Z1M13 <SEP> Primer <SEP> (JOE) <SEP> 1 <SEP> L <SEP> 1 <SEP> L
<tb> DTTP <SEP> mix <SEP> 1 <SEP> L <SEP> 1 <SEP> L
<tb> Morpholino <SEP> T <SEP> glycine <SEP> 2 <SEP> mM <SEP> 1 <SEP> L <SEP> 2.5 <SEP> uL
<tb> Taq <SEP> (3U / L) <SEP> 1 <SEP> L <SEP> 1 <SEP> L
<tb> Matrix <SEP> 1 <SEP> L <SEP> 1 <SEP> L
<tb> H20 <SEP> 3 <SEP> L <SEP> 1.5 <SEP> L
<tb>

<Desc / Clms Page number 27>

<tb>
<tb> Table <SEP> 5
<tb> ddTTP <SEP> 250 <SEP> M <SEP> ddTTP <SEP> 300 <SEP> M
<tb> Buffer <SEP> TACS <SEP> (x5) <SEP> 2 <SEP> L <SEP> 2 <SEP> \ il
<tb> Z1 <SEP> M <SEP> 13 <SEP> Primer <SEP> (ROX) <SEP> 1 <SEP>, iL <SEP> 1 <SEP> L
<tb> DTTP <SEP> mix <SEP> 1 <SEP> L <SEP> 1 <SEP> L
<tb> DdTTP <SEP> 2.5 <SEP> mM <SEP> 1 <SEP> L <SEP> 2.5 <SEP> L
<tb> Taq <SEP> (3U / L) <SEP> 1 <SEP> L <SEP> 1 <SEP> L
<tb> Matrix <SEP> 1 <SEP> L <SEP> 1 <SEP> L
<tb> H20 <SEP> 3 <SEP> L <SEP> 1.5 <SEP> L
<tb>

The products of the sequence reactions are detected by fluorescence. The results obtained are represented in the appended figure which illustrates the detection of the products in the sequencing gel analyzed by the Perkin Elmer Analysis software, version 3.0.

For each test, the primers are identifiable by their fluorescence properties, the ROX marker (red) for the control reaction with dideoxythymidine triphosphate 250 m (dashed curve) and the JOE marker (green) for the reaction concerning morpholino T glycine 200 m (solid line curve).

As shown in the figure, the results of these tests are quite conclusive since the morpholino T glycine is well incorporated in a base specific manner by the Taq polymerase, and acts well as a chain terminator.

The other three morpholino-nucleotides can be used in the same way to determine the positions of the four bases of DNA in the fragment to be analyzed.

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Example 6 Test of morpholino A putrescine and morpholino A fluorescein in sequencing
Morpholino A putrescine (MATPP) and morpholino A fluorescein (MATPPF) are tested in sequence reaction with fluorescent primers (Applied Biosystems, Perkin-Elmer, Foster City, CA, USA) on a standard matrix which is Bluescript plasmid DNA ( Stratagene, La Jolla, CA, USA). The enzyme used is a Taq Polymerase (Perkin-Elmer) which is used in its buffer (Thermo Sequenase buffer, Amersham, Life Science).

Three sequence reactions were carried out with MATPP at 100,200 and 400 M and four sequence reactions with MATPPF at 200,500, 1000 and 5000 M, as well as control reactions with the dideoxynucleotide ddATP at the concentration of 250 µM (Boehringer).

The reaction medium with a total volume of 10 µL contains 125 ng of template, 1.25 pmoles of fluorescent primer and the other constituents as described in the tables.

The medium is subjected to thermal cycles in order to produce a number of molecules of newly formed DNA strands. Amplification on a Perkin-Elmer 9700 apparatus (Gene Amp #, PCR System 9700) is carried out, according to the following sequences: MATPP 3 min, 95 C; 30 cycles (15 sec, 95 C; 15 sec, 55 C; 1 min, 70 C) MATPPF 3 min, 95 C; 30 cycles (15 sec., 95 C; 15 sec, 55 C; 4 min, 60 C)
The amplification products are purified on a Sephadex G50 column. The products of each sequence reaction are mixed with the products of a control reaction and analyzed by electrophoresis.

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The mixture obtained is migrated in a denaturing gel (7M urea) of acrylamide of the Long Ranger type (6%), in TBE IX, on an Applied Biosystems 377 apparatus (ABI Prism DNA Sequencer, Perkin Elmer).

Electrophoresis takes place for 7 hours at 1680 V, 50 mA.

Preparation of the nucleotide stock solution: dATP mix for 16 reactions here representing a mixture of four natural nucleotide triphosphates, depleted in deoxyadenosine triphosphate (called dATP mix): 4 L of a 1.25 mM solution of dATP (Promega) are mixed. with 4 (iL 5mM dTTP (Promega), 4L 5mM dCTP (Promega) 4L 5mM dGTP (Promega).

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Table 6
Preparation of the common Mix for 15 reactions / reaction / 15 reactions
TACS buffer (x5) 2 L 30 L dATP mix 1 L 15 L
Taq (5U / L) 1 L 15 L
Matrix (plasmid 2 L 30 L
Bluescript) Preparation of the 2 mM stock solution of MATPP: (1.17 mg of MATPP is diluted in 1.04 ml of H2O.

Table 7
Reactions with 2mM morpholino ATPP

<tb>
<tb> Morpholino <SEP> Morpholino <SEP> Morpholino
<tb> ATPP400M <SEP> ATTP <SEP> 200 <SEP> M <SEP> ATTP <SEP> 100 <SEP> M
<tb> Mix <SEP> common <SEP> 6 <SEP> L <SEP> 6 <SEP> \ iL <SEP> 6 <SEP> L
<tb> Z1M13 <SEP> Primer <SEP> (JOE) <SEP> 1 <SEP> L <SEP> 1 <SEP> L <SEP> 1 <SEP> L
<tb> Morpholino <SEP> ATPP <SEP> 2 <SEP> mM <SEP> 2 <SEP> L <SEP> 1 <SEP> L <SEP> 0.5 <SEP> (iL
<tb> H20 <SEP> 1 <SEP> L <SEP> 2 <SEP> L <SEP> 2.5 <SEP> pL <SEP>
<tb>

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Table 8 Three control reactions with 2.5 mM dideoxyadenosine triphosphate (ddATP)

<tb>
<tb> DdATP <SEP> 250 <SEP> M
<tb> Mix <SEP> common <SEP> 6 <SEP> L
<tb> Z1M13 <SEP> Primer <SEP> (ROX) <SEP> 1 <SEP> L
<tb> DdATP <SEP> 2.5 <SEP> mM <SEP> 1 <SEP> L
<tb> H20 <SEP> 2 <SEP> L
<tb>
Preparation of 20 mM and 2 mM stock solutions of MATPPF 20 mM So solution: dilute the sample (2.2 mg) in 110.5 μL of H2O 2 mM Si solution: take 10 L of So and add 90 L of 'H20
Table 9
Reactions with 20 mM ATPPF morpholino (So)

<tb>
<tb> MATPPF <SEP> 1000 <SEP> M <SEP> MATTPF <SEP> 5000 <SEP> M
<tb> Mix <SEP> common <SEP> 6 <SEP> L <SEP> 6 <SEP> L
<tb> Z1M13 <SEP> Primer <SEP> (JOE) <SEP> 1 <SEP> L <SEP> 1 <SEP> L
<tb> Morpholino <SEP> ATPPF <SEP> 20 <SEP> mM <SEP> 0.5 <SEP> L <SEP> 2.5 <SEP> L
<tb> H2O <SEP> 2.5 <SEP> L <SEP> 0, <SEP> 5 <SEP> (iL <SEP>
<tb>

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Table 10 Reactions with 2 mM morpholino ATPPF (Si)

<tb>
<tb> MATPPF <SEP> 500 <SEP> M <SEP> MATTPF <SEP> 200 <SEP> M
<tb> Mix <SEP> common <SEP> 6 <SEP> L <SEP> 6 <SEP> L
<tb> Z1M13 <SEP> Primer <SEP> (JOE) <SEP> 1 <SEP> L <SEP> 1 <SEP> L
<tb> Morpholino <SEP> ATPPF <SEP> 2 <SEP> mM <SEP> 2.5 <SEP> L <SEP> 1 <SEP> L
<tb> H2O <SEP> 0.5 <SEP> (iL <SEP> 2 <SEP> (iL <SEP>
<tb>

Table 11 Four control reactions with 2.5 mM dideoxyadenosine triphosphate (ddATP)

<tb>
<tb> ddATP <SEP> 250 <SEP> M
<tb> Mix <SEP> common <SEP> 6 <SEP> L
<tb> Z1M13 <SEP> Primer <SEP> (ROX) <SEP> 1 <SEP> L
<tb> ddATP <SEP> 2.5 <SEP> mM <SEP> 1 <SEP> L
<tb> H2O <SEP> 2 <SEP> (iL
<tb>

The results obtained with morpholino A putrescine at 100 μM and morpholino A fluorescein at 5 mM, between the 90th and the 250th base, are given in FIG. 2.

It can therefore be seen that these two derivatives act well as chain terminators. In addition, it should be noted that the reactions carried out with the fluorescent derivative, morpholino A fluorescein, were detected by the fluorophore carried by this derivative: a fluorescent chain terminator was therefore prepared.

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Example 7: Use of morpholino A putrescine (MATPP) and morpholino A fluorescein (MATPPF) for the matrix-dependent labeling of DNA fragments in 3 '; test of the enzymatic incorporation of these compounds by three polymerases (Taq, Klenow, Klenow Exo Free) and a reverse transcriptase.

These two nucleoside triphosphate derivatives are tested by enzymatic incorporation to label an oligonucleotide 13 bases long at its 3 'end.

This labeling is said to be “dependent matrix” because the enzymes used need the complementary target in order to lengthen the oligonucleotide according to the rules of Watson & Crick. The sequence A (17870 pmol / mL) studied as well as its target C (16128 pmol / mL) are given in the figure below: Target C: 3'-TGC CAA CCA ACC CCA CCT CAA CCT CTG-5 'Primer A: 5 '-ACG GTT GGT TGG G (13 bp) Expected fragments: 5'-ACG GTT TGG GGT GGA (18 bp) and lengths (bp): 5'-ACG GTT GGT TGG GGT GGA GTT GGA (24 bp)
5'-ACG GTT GGT TGG GGT GGA GTT GGA GA (26 bp)
5'-ACG GTT GGT TGG GGT GGA GTT GGA GAC (27 bp)
Three enzymes are used for this labeling: Taq DNA polymerase (Boehringer Mannheim), Klenow (Boehringer Mannheim) and Klenow Exonuclease Free (Amersham Life Science). The primer is labeled at its 5 ′ end by incorporation of 32P phosphate with the “Ready to go” T4 Polynucleotide Kinase kit (Pharmacia Biotech). The radiolabeled primer is denoted A *.

Reaction buffers of the three enzymes are prepared for 10 reactions:

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Table 12

<tb>
<tb> (in <SEP> L) <SEP> Reaction <SEP> Taq <SEP> Reaction
<tb> Klenow <SEP> Exo
<tb> Free
<tb> C <SEP> 50 <SEP> 50
<tb> A <SEP> 10 <SEP> 10
<tb> A * <SEP> 10 <SEP> 10
<tb> Tp <SEP> 10X <SEP> 50 <SEP> 50
<tb> H20 <SEP> 50 <SEP> 50
<tb>
Table 13

<tb>
<tb> (in <SEP> L) <SEP> Reaction
<tb> ~~~~~~~~~~~~ <SEP> Klenow
<tb> C <SEP> 50
<tb> A <SEP> 10
<tb> A * <SEP> 10
<tb> Tp <SEP> 5X <SEP> 100
<tb> H2O <SEP> 0
<tb>

The enzymes are then diluted as follows, for 10 reactions: - Taq (5U / pL); 10X0.1 L of Taq + 10X15.5 pL of H2O - Klenow (20U / L): 10X0.1 uL of Klenow + 10X15.5 uL of H2O - Klenow Exo Free (5U / uL): 10X0.1 L of Klenow Exo Free + 10X15.5 pL of H2O-
Solutions containing the normal nucleoside triphosphates are also prepared: - "2P" solution composed of a mixture of dGTP and dTTP at
0.1 mM each - "4P" solution composed of a mixture of dATP, dCTP, dGTP and dTTP at 0.1 mM each
The implementation reactions are described in Table 14 below:

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Table 14

<tb> (in <SEP> L) <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9
<tb> 400 <SEP> M <SEP> 200 <SEP> M <SEP> 50 <SEP> M <SEP> 2.5 <SEP> mM <SEP> 400 <SEP> M <SEP> 200 <SEP> M <SEP> 50 <SEP> M
<tb> Reaction <SEP> 17 <SEP> 17 <SEP> 17 <SEP> 17 <SEP> 17 <SEP> 17 <SEP> 17 <SEP> 17 <SEP> 17 <SEP>
<tb>"2P"<SEP> 0 <SEP> 5 <SEP> 5 <SEP> 5 <SEP> 5 <SEP> 5 <SEP> 5 <SEP> 5 <SEP> 0 <SEP>
<tb>"4P"<SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 5
<tb> MATPP <SEP> 2 <SEP> mM <SEP> 0 <SEP> 10 <SEP> 5 <SEP> 1.25 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP>
<tb> MATPPF <SEP> 20 <SEP> mM <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 6.25 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0
<tb> MATPPF <SEP> 2mM <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 10 <SEP> 5 <SEP> 1.25 <SEP> 0
<tb> H2O <SEP> 33 <SEP> 2.7 <SEP> 7.4 <SEP> 11.15 <SEP> 6.15 <SEP> 2.4 <SEP> 7.4 <SEP> 11.15 <SEP> 12.4
<tb> Enzyme <SEP> 0 <SEP> 15.6 <SEP> 15.6 <SEP> 15.6 <SEP> 15.6 <SEP> 15.6 <SEP> 15.6 <SEP> 15.6 <SEP> 15.6
<tb>

<Desc / Clms Page number 36>

The putrescine morpholino A is thus tested at three concentrations: 400,200 and 50 M while the fluorescein morpholino A is reacted with
2.5 mM, 400,200 and 50 M.

Before adding the enzyme, the mixture is denatured at 94 ° C. for 5 minutes. Then allowed to return to room temperature so that hybridization takes place. Elongation is carried out at 70 C for the
Taq, 37 C for both Klenow, and this during
10 minutes. Finally, the medium is again denatured with a solution of formamide and heating at 90 ° C. for
5 minutes before being deposited on a polyacrylamide gel. The separation is carried out by electrophoresis at 2000 V. The gel is read at
Phosphorimager; the results obtained are shown in figure 3.

In this figure, lanes number 1 serve as a migration control for the labeled oligonucleotide A. This oligonucleotide is 13 bases (13-mer) in length. Lanes 2, 3 and 4 make it possible to follow the elongation of oligonucleotide A and the incorporation of morpholino A putrescine. Under these conditions, only the dGTP and dTTP nucleotides (“2P” solution) were added and can be used by the enzyme to carry out the extension of the primer. The presence of morpholino A putrescine in the reaction medium allows its incorporation at the level of the base 18. A control was carried out, by placing in the medium only the “2P” mixture; in this case, the enzyme continues its extension to the 17th base since it does not have an adenosine derivative to continue its polymerization. Thus, the difference in migration between this control, 17 bases long, and reactions 2, 3 and 4 confirms

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incorporation of MATPP and interruption of chain elongation. Reactions 5 to
8 correspond to the same reactions with morpholino
A fluorescein. Here again, the MATPPF is well incorporated and stops the polymerization of the complementary strand. It is noted, however, for the two Klenows, that there has sometimes been incorporation of another base (G or T) in place of the morpholino derivative. In fact, in these cases, elongation products are found, corresponding to
18- and 24-Wed.

Well 9 (see figure 4) is a control reaction: the reaction medium contains the
4 normal deoxynucleotides and can therefore extend the primer to its maximum extension, i.e. until 27-mer is obtained.

In conclusion, the three enzymes incorporate morpholino A putrescine and morpholino A fluorescein in all the concentrations tested, including at the lowest concentrations.

The ability of reverse transcriptases to incorporate morpholinonucleotide derivatives during oligonucleotide extension has been confirmed. In this test, reverse transcriptase (M-MLV, Promega; activity: 200,000 U / mL) is chosen as a model.

The latter is capable of synthesizing a DNA strand complementary to a target (DNA or RNA), from an oligonucleotide primer, in the presence of nucleoside triphosphates. The putrescine morpholino A and fluorescein morpholino A are therefore tested at final concentrations of 250 M. A control copy is also deposited on the gel, with the four nucleoside triphosphates of the “4P” solution.

<Desc / Clms Page number 38>

The sequence of target C (27-mer, 16128 pmol / mL) is that of primer B (14-mer, 56368 pmol / mL) are shown below. This radioactively labeled B primer is denoted B *.

Solution B * then contains 10 pmol of primer B in a volume of 50 µL. The solutions of C and B are also diluted ten times; these solutions are noted respectively C / 10 and B / 10.

Target C: 3'-TGC CAA CCA ACC CCA CCT CAA CCT CTG-5 'Primer B: 5'-ACG GTT GGT TGG GG (14 bp)
Table 15

<tb>
<tb> (in <SEP> L) <SEP> Reaction <SEP> Reaction <SEP> Reaction <SEP> Reaction
<tb> 1 <SEP> 2 <SEP> 3 <SEP> 4
<tb> C / 10 <SEP> 2 <SEP> 2 <SEP> 2 <SEP> 0
<tb> B * <SEP> 5 <SEP> 5 <SEP> 5 <SEP> 5
<tb> B / 10 <SEP> 3 <SEP> 3 <SEP> 3 <SEP> 0
<tb> Buffer <SEP> 5X <SEP> 4 <SEP> 4 <SEP> 4 <SEP> 0
<tb> MATPP <SEP> 2 <SEP> mM <SEP> 2.5 <SEP> 0 <SEP> 0 <SEP> 0
<tb> MATPPF <SEP> 2 <SEP> mM <SEP> 0 <SEP> 2.5 <SEP> 0 <SEP> 0
<tb>"2P"<SEP> 2.5 <SEP> 2.5 <SEP> 0 <SEP> 0
<tb>"4P"<SEP> 0 <SEP> 0 <SEP> 2.5 <SEP> 0
<tb> H2O <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 15
<tb> Enzyme <SEP> 1 <SEP> 1 <SEP> 1 <SEP> 0
<tb>

As previously, the mixture is denatured, before the addition of the enzyme, at 94 ° C. for 5 minutes and the mixture is allowed to return to room temperature. Elongation takes place at 37 C for 60 minutes. The medium is denatured with a formamide solution and heated at 90 ° C. for 5 minutes before being deposited on a polyacrylamide gel. The separation is carried out by electrophoresis at 1500 V. The gel is read at

<Desc / Clms Page number 39>

Phosphorimager; the results obtained are given in figure 4.

In this figure, track 4 makes it possible to estimate the length of the marked primer B. Lane 3 shows the maximum extension of primer B to a final product of 27 base pairs in the presence of the 4 natural deoxynucleotides. Reactions 1 and 2 show that the morpholino derivatives are incorporated during the extension of primer B by reverse transcriptase. This incorporation is quantitative and provides a product of 18 base pairs (in the absence of a morpholino derivative, the extension is blocked at the 17th base).

In conclusion, the morpholino derivatives are very well recognized by reverse transcriptase and incorporated into the primers during extension according to a specific base process.

References cited [1]: Sanger et al, Proceedings of National Academy of
Science, 74,1977, p. 5463-5467.

[2]: WO-A-96/23807.

[3]: Prober et al, Science, 238,1987, pages 336-341.

[4]: Hileman et al, Bioconjugate Chemistry, 5.1994, pages 436-444.

[5]: Broker et al, Nucleic Acids Research, 5.1978, pages 363-385.

[6]: Agrawal et al, Nucleic Acids Research, 14,1986, pages 6227-6245.

[7]: FR-A-2 710 068 [8]: Rayford et al, Journal of Biological Chemistry,
260,1985, pages 15708-15713.

Claims (15)

1. Process for the manufacture of a nucleic acid fragment (DNA or RNA) labeled 3 ', which comprises the enzymatic incorporation of a nucleotide derivative having as a precursor a compound of formula:

in which R represents a nucleic base and R represents a group corresponding to one of the following formulas: - (CH2) n-NH2 - (CH2) n-SH - (CH2) n-COOH - (CH2) n-OH - (CH2) n-NH-R3 - (CH2) n-SR3 - (CH2) n-CO-R3 - (CH2) n-OR 3 where n is an integer ranging from 1 to 12 and R3 is a derived group a label, a protein, an enzyme, a fatty acid or a peptide, at the 3 'OH end of the nucleic acid fragment. 2. Process for modifying a nucleic acid fragment by enzymatic incorporation into 3 'of a modified morpholino-nucleotide having as a precursor a compound corresponding to the formula: <Desc / Clms Page number 41> in which R1 represents a nucleic base and R2 represents a group corresponding to one of the following formulas: - (CH2) n-NH-R3 - (CH2) n-CO-R3 - (CH2) n-SR3 - (CH2) n-OR3 in which R represents a compound chosen from photocrosslinking agents, fatty acids, hydrophobic peptides, antibodies, enzymes and fluorophores.

3. A method of sequencing a nucleic acid (DNA or RNA) by the technique of enzymatic polymerization of the complementary sequence of this nucleic acid using chain terminators, in which at least one of the chain terminators has for precursor a compound corresponding to the formula:

<Desc / Clms Page number 42> in which R1 represents a nucleic base and R2 represents a group corresponding to one of the following formulas: - (CH2) n-NH2 - (CH2) n-SH - (CH2) n-COOH - (CH2) n-OH - (CH2) n-NH-R3 - (CH2) n-SR '- (CH2) n-CO-R3 - (CH2) n-OR3 in which n is an integer ranging from 1 to 12 and R is a derived group a label, a protein, an enzyme, a fatty acid or a peptide. 4. The method of claim 1, 2 or 3, wherein the enzyme is the Klenow fragment of DNA polymerase. 5. The method of claim 1, 2 or 3, wherein the enzyme is a heat-resistant polymerase of a thermophilic bacterium or terminal transferase or reverse transcriptase. 6. Method according to any one of claims 1 to 5, in which the nucleic acid base is a natural nucleic acid base chosen from adenine, guanine, cytosine, thymine, uracil, xanthine, hypoxanthine, 2-aminopurine and their derivatives. 7. Method according to any one of claims 1 to 5, in which R1 corresponds to one of the following formulas: <Desc / Clms Page number 43>

8. Method according to any one of claims 1 to 7, wherein the marker is chosen from radioactive products, luminescent, electroluminescent and fluorescent products, molecules capable of coupling to other molecules, molecules allowing interactions. of the antigen-antibody type, and enzymatic markers. 9. A method according to any one of claims 1 to 7, wherein R3 is a fluorophore. 10. The method of claim 9, wherein R3 is chosen from derivatives of fluorescein, biotin and rhodamine. 11. The method of claim 1, 2 or 3 wherein the derivative, the modified morpholino-nucleotide, or the chain terminator is compound (I) in the form of monophosphate. 12. Morpholino-nucleotide corresponding to the formula: <Desc / Clms Page number 44> wherein R1 is adenine and R2 represents -CH2-COOH, - (CH2) 4-NH2 or - (CH2) 4-NH-R3 with R3 representing a group derived from fluorescein.

13. Morpholino-nucleotide of formula:

wherein R1 is thymine and R2 is -CH2COOH. 14. A method of manufacturing a morpholinonucleotide of formula:

<Desc / Clms Page number 45> in which R1 has the meaning given above, with a periodate, a compound of formula RNH2 in which R2 has the meaning given above and sodium borohydride.

in which R1 represents a nucleic base and R represents a group corresponding to one of the following formulas: - (CH2) n-NH2 - (CH2) n-SH - (CH2) n-COOH - (CH2) n-OH - (CH2) n-NH-R3 - (CH2) n-SR3 - (CH2) n-CO-R3 - (CH2) n-OR3 in which n is an integer ranging from 1 to 12 and R3 is a group derived from d 'a marker, of a protein, of an enzyme, of a fatty acid or of a peptide, the reaction of a nucleoside triphosphate of formula: 15. Use of a morpholino-nucleotide of the formula:

<Desc / Clms Page number 46> in which R1 represents a nucleic base and R represents a group corresponding to one of the following formulas: - (CH2) n-NH2 - (CH2) n-SH - (CH2) n-COOH - (CH2) n-OH - (CH2) n-NH-R3 - (CH2) n-SR3 - (CH2) n-CO-R3 - (CH2) n-OR3 in which n is an integer ranging from 1 to 12 and R3 is a group derived from d 'a marker, of a protein, of an enzyme, of a fatty acid or of a peptide, for the labeling of fragments of DNA or of RNA.