WO2001042495A2 - Produits comprenant un support sur lequel sont fixes des acides nucleiques et leur utilisation comme puce a adn - Google Patents
Produits comprenant un support sur lequel sont fixes des acides nucleiques et leur utilisation comme puce a adn Download PDFInfo
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- WO2001042495A2 WO2001042495A2 PCT/FR2000/003427 FR0003427W WO0142495A2 WO 2001042495 A2 WO2001042495 A2 WO 2001042495A2 FR 0003427 W FR0003427 W FR 0003427W WO 0142495 A2 WO0142495 A2 WO 0142495A2
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- Prior art keywords
- group
- support
- oligonucleotide
- function
- oxoaldehyde
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- 0 *C1*C(*N)SC1 Chemical compound *C1*C(*N)SC1 0.000 description 3
- BNRQFWDDZOTSIL-UHFFFAOYSA-N CC(C)CSCN Chemical compound CC(C)CSCN BNRQFWDDZOTSIL-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00497—Features relating to the solid phase supports
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00497—Features relating to the solid phase supports
- B01J2219/00527—Sheets
- B01J2219/00529—DNA chips
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/0059—Sequential processes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00596—Solid-phase processes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
- B01J2219/00608—DNA chips
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
- B01J2219/0061—The surface being organic
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
- B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
- B01J2219/00614—Delimitation of the attachment areas
- B01J2219/00617—Delimitation of the attachment areas by chemical means
- B01J2219/00619—Delimitation of the attachment areas by chemical means using hydrophilic or hydrophobic regions
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
- B01J2219/00623—Immobilisation or binding
- B01J2219/00626—Covalent
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00659—Two-dimensional arrays
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/0068—Means for controlling the apparatus of the process
- B01J2219/00693—Means for quality control
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/0068—Means for controlling the apparatus of the process
- B01J2219/00702—Processes involving means for analysing and characterising the products
- B01J2219/00707—Processes involving means for analysing and characterising the products separated from the reactor apparatus
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00718—Type of compounds synthesised
- B01J2219/0072—Organic compounds
- B01J2219/00722—Nucleotides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/11—Compounds covalently bound to a solid support
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
- C12Q2565/50—Detection characterised by immobilisation to a surface
- C12Q2565/501—Detection characterised by immobilisation to a surface being an array of oligonucleotides
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
Definitions
- the present invention relates to products comprising a support on which nucleic acids are fixed, to their preparation process and to their use as DNA chips.
- the present invention also relates to functionalized supports, to oligonucleotides and to DNAs modified in the 5 ′ position, as well as to their methods of preparation.
- the present invention further relates to a method of attaching a nucleic acid to a support.
- the process for obtaining DNA chips which implements DNA fixation on a glass slide mainly involves steps for preparing the glass slide (treatment of its surface with sodium hydroxide, then adsorption of polylysine or polyethyleneimine on the surface via ionic interactions; BURNS, NL, Langmuir, 1995, il, 2768-2776), depositing DNA on the glass slides thus prepared, then heat treatment and UN irradiation to bind , covalently, the AD ⁇ on the glass surface.
- DNA normally makes it possible to obtain very specific hybridizations and of high affinity compared to that which is obtained with relatively short oligonucleotides.
- the method of manufacturing the blades can explain this phenomenon.
- DNA interacts with polylysine or polyethyleneimine by ionic interactions involving the negatively charged phosphodiester groups of nucleic acids: this mode of deposition thus limits the conformational freedom of DNA and its accessibility.
- the heat treatment applied before UV irradiation degrades the DNA.
- UV irradiation itself modifies the pyrimidine bases (formation of hydrates or binding to the surface via the silanol functions of the glass, dimerization between several pyrimidines) while these bases are important for hybridization.
- US Pat. No. 4,874,813 describes the fixing of glycoproteins on a solid support by a hydrazone bond, the support being functionalized by a hydrazide and the glycoprotein carrying an aldehyde function, introduced by oxidation of the carbohydrate part of the glycoprotein, carrying a 1,2-diol function.
- this technique cannot be extrapolated to DNAs, which are naturally devoid of 1,2-diol functions, or to any nucleic acids.
- the glycoproteins used in US Pat. No. 4,874,813 are obtained with quantities of the order of ten milligrams, the nucleic acids are manipulated on the microgram scale.
- nucleic acid carrying an aldehyde function at its end is not very stable, in particular subject to oxidation reactions in the presence of air, and easily gives rise to the formation of imines when it is put in the presence of enzymes. , for example during amplification reactions.
- the inventors have given themselves the aim of overcoming the drawbacks of the prior art and of providing a method for fixing nucleic acids, in particular DNA or oligonucleotides, on a support which meets in particular the following criteria: - the process is simple at the experimental level, reproducible and inexpensive,
- the subject of the present invention is a product of formula (I):
- Z represents a group of formula
- a group -XN CH-, X representing a group -CH 2 -O-, -CH 2 -NH- or -NH-, i is equal to 0 or to 1, n is between 1 and 16, n being equal to 1 when i is equal to O, m is greater than or equal to 1, SP represents a support, A represents a spacer arm,
- Y represents a function ensuring the connection between A and Z
- nucleic acid means a DNA, an RNA or an oligonucleotide, the latter corresponding to a sequence of approximately 1 to 50 bases, said nucleic acid comprising natural nucleosides (A, C, G , T or U) or modified at the base (heterocycle), sugar and / or phosphodiester bond.
- Said nucleic acid can therefore also consist of a PNA (Peptide Nucleic Acid).
- the product of formula (I) can also comprise a thiazolidine bond when Z represents a group of formula:
- the product of formula (I) according to the present invention can therefore correspond to the products of formulas (la) and (Ib) represented below, in which SP, A, Y, X, M, i , n and m are as previously described:
- the product of formula (I) according to the present invention is such that the binding between nucleic acid M and the support SP is very stable.
- n is advantageously equal to 1 and A preferably represents a linear or branched carbon chain comprising from 2 to 100 carbon atoms, preferably from 5 to 50 carbon atoms, and optionally comprising from 1 to 35 oxygen or nitrogen atoms and from 1 to 5 silicon, sulfur or phosphorus atoms.
- SP advantageously represents a solid support, preferably made of glass, silicon or synthetic polymer, such as nylon, polypropylene and polycarbonate.
- SP can also advantageously represent a non-solid support, such as a natural polymer (for example a polysaccharide such as cellulose or mannan, or even a polypeptide), a synthetic polymer (for example a copolymer of N- vinylpyrrolidone and derivatives of acrylic acid), a liposome or a lipid.
- SP can advantageously represent a transfection vector, that is to say an organic compound (lipid or peptide for example) permeable at the level of the cell membrane.
- SP is a solid support, i is equal to 1, n is equal to 1 and M is DNA, said product constituting a DNA chip.
- the m molecules M present in the chip can be identical, but are preferably different from each other.
- the DNA chips according to the present invention are reusable in numerous hybridization cycles, the bond between the DNA and the support being very stable under the hybridization and washing conditions, which considerably limits the possibilities of desorption of the DNA.
- SP represents a glass support
- i is equal to 1
- n is equal to 1
- A represents a spacer arm of formula -Si- (CH 2 ) 3 - and Y represents an amide function -NH-CO-.
- the present invention also relates to the use of the product of formula (I) above, when SP is a solid support, as a nucleic acid chip, such as a DNA or oligonucleotide chip.
- the present invention also relates to a process for the preparation of the product of formula (I) above, which comprises the reaction of nxm molecules of formula M-CO-CHO with a product of formula SP [A, (Y, - B - NH 2 ) n ] m , SP, A, Y, i, n, m and M being as defined above and B representing a group -CH 2 -O-, -CH 2 -NH-, -NH- or -CH (CH 2 SH) -.
- the molecules of formula M-CO-CHO correspond to nucleic acids carrying an ⁇ -oxoaldehyde (-CO-CHO) function at their 5 ′ or 3 ′ end.
- the present invention also relates to a process for fixing, by covalent bond, of at least one nucleic acid M on an SP support, for obtaining a product of formula (I) as previously described, characterized in that that it comprises the following steps: i) introduction of an ⁇ -oxoaldehyde function at a 5 ′ or 3 ′ end end of said nucleic acid, and ii) reaction of the functionalized nucleic acid obtained in step i) with a support modified by a function selected from the group consisting of hydrazine functions, derived from hydrazine, hydroxylamine and ⁇ -aminothiol.
- hydrazide functions that is to say a hydrazine substituted with at least one acyl or carbonyl group.
- the method according to the present invention implements a support modified by a stable function in a wide pH range.
- the functions involved in the binding namely the ⁇ -oxoaldehyde function carried by the nucleic acid and the function carried by the support, are very reactive.
- the process according to the present invention does not involve any denaturation of the structure of the nucleic acid, which remains optimal for subsequent hybridization reactions, in the case where the product of formula (I) obtained by the process according to the present invention is used as a DNA chip.
- the introduction of an ⁇ -oxoaldehyde function at one end of said nucleic acid is carried out by the following steps: a) introduction of a group selected from the group consisting with tartaric acid, serine, threonine and their derivatives at one of the ends of an oligonucleotide, b) hybridization of the oligonucleotide obtained in step a) with said nucleic acid, c) elongation of said oligonucleotide, d) repeating steps b) and c) at least once, e) periodic oxidation of the nucleic acid obtained in step d), modified at one of its ends by a group selected from the group consisting of tartaric acid, serine, threonine and their derivatives, and f ) isolation of a nucleic acid modified at one of its ends by an ⁇ -oxoaldehyde function.
- the introduction of an ⁇ -oxoaldehyde function at one end of said nucleic acid is carried out by the following steps: a) introduction of a group selected from the group consisting of tartaric acid, serine, threonine and their derivatives at one end of an oligonucleotide, b) periodic oxidation of the oligonucleotide obtained in step a), c) hybridization of the oligonucleotide obtained in step b), carrying an ⁇ -oxoaldehyde function at one of its ends, with said nucleic acid, d) elongation of said oligonucleotide, e) repeating steps c) and d) at least once, and f) isolation of a nucleic acid modified at one of its ends by an ⁇ -oxoaldehyde function.
- the step of hybridizing the oligonucleotide with the nucleic acid is carried out after a step of denaturing said nucleic acid, as known to those skilled in the art.
- the stages of hybridization between an oligonucleotide and a nucleic acid, of elongation of said oligonucleotide and of repeating these stages constitute amplification cycles of the nucleic acid, the oligonucleotide serving as a primer for these amplifications, which can be performed by the technique known as PCR (Polymerase Chain Reaction) well known to those skilled in the art, described for example in Molecular Cloning, second edition, J. SAMBROOK, EF FRITSCH and T. MANIATIS (Cold Spring Harbor Laboratory Press, 1989).
- PCR Polymerase Chain Reaction
- the elongation step of the oligonucleotide, after its hybridization with the nucleic acid, is carried out in a suitable buffer medium, in the presence of the nucleotide bases required for the formation of nucleic acid.
- tartaric acid derivatives which can be used in the processes described above, mention may be made of di-acetyl-tartaric acid, di-para-toluyl-tartaric acid, metatartaric acid, dimethyl tartrate, dissuccinimidyl tartrate, tartaric anhydride and di-acetyl-tartaric anhydride.
- the introduction of a selected group in the group consisting of tartaric acid, serine, threonine and their derivatives at one of the ends of an oligonucleotide is carried out via an amide bond.
- the group selected from the group consisting of tartaric acid, serine, threonine and their derivatives is preferably linked to the oligonucleotide via a spacer arm linked, by one of its ends, to said oligonucleotide and carrying, to the 'other of its ends, an amino function.
- the nucleic acid is preferably DNA.
- the oligonucleotide which hybridizes with this nucleic acid is an oligodeoxynucleotide primer, which can be specific or universal.
- DNA can be obtained by amplification of genomic DNA or by amplification of DNA inserted into a vector, for example phage M13.
- a first amplification with a series of specific primers can be followed by amplification using universal primers (see for example POLLACK, JR in Nature Genetics, 1999, 21, 41-46). In this case, two primers make it possible to amplify a large number of different DNAs.
- the present invention also relates to an oligonucleotide modified in position 5 ′ by a group selected from the group consisting of tartaric acid, serine, threonine, their derivatives and the ⁇ -oxoaldehyde group.
- Such an oligonucleotide can advantageously be used as a primer in reactions for elongation or amplification of nucleic acids, in order to obtain nucleic acids modified in position 5 ′ by the groups carried by said oligonucleotide.
- the present invention also relates to a process for the preparation of such an oligonucleotide, characterized in that it comprises a step of introduction of a group selected from the group consisting of tartaric acid, serine, threonine and their derivatives in position 5 ′ of said oligonucleotide, this step being followed, in the case where said oligonucleotide is modified by an ⁇ -oxoaldehyde group, of periodic oxidation of said oligonucleotide.
- the present invention also relates to a DNA modified in the 5 ′ position by a group selected from the group consisting of tartaric acid, serine, threonine, their derivatives and the ⁇ -oxoaldehyde group.
- Such DNA can advantageously be used in the method according to the present invention allowing the attachment, by covalent bond, of at least one nucleic acid M on an SP support, as described above.
- the tartaric acid, serine and threonine groups can easily be converted to the ⁇ -oxoaldehyde function by a periodic oxidation reaction. It is particularly advantageous to use DNA modified by an ⁇ -oxoaldehyde function, because this function is very stable and very reactive, in any event much more stable and reactive than an aldehyde function. These considerations also apply to the oligonucleotides according to the present invention, modified by an ⁇ -oxoaldehyde function. Thus, whatever nucleic acids (notably DNA or oligonucleotides) can be preserved without oxidizing or degrading, in particular in the presence of air (regardless of the position of their functionalization at the 3 ′ end or 5 ').
- nucleic acids functionalized by an aldehyde they give rise to very stable bonds with supports carrying corresponding reactive functions, as described above.
- they do not induce imine formation with the enzymes present during reactions of elongation or amplification of nucleic acids and, in the case of a PCR amplification using Taq polymerase, they do not interact with dithiothreitol, the enzyme's preservative.
- the present invention also relates to a process for the preparation of a DNA modified in the 5 ′ position by a group selected from the group consisting of tartaric acid, serine, threonine, their derivatives and the ⁇ -oxoaldehyde group, such as described above, characterized in that it comprises the following steps: a) introduction of a group selected from the group consisting of tartaric acid, serine, threonine and their derivatives in position 5 'of a oligonucleotide, b) hybridization of the oligonucleotide obtained in step a) with DNA, c) elongation of said oligonucleotide, d) repetition of steps b) and c) at least once, or in the case where said DNA is modified by an ⁇ -oxoaldehyde group, steps a) to f) of the methods previously described in relation to the method according to the present invention relating to the introduction of a function ⁇ -oxoalde
- the elongation step of the oligonucleotide, after its hybridization with DNA, is carried out in a suitable buffer medium, in the presence of the deoxynucleotide bases required for the formation of DNA.
- the present invention also relates to a functionalized support of formula (II):
- Such a support carries a hydrazine function (case where B represents a group -CH 2 -NH- or -NH-), hydroxylamine (case where B represents a group -CTL-O-) or ⁇ -aminothiol (case where B represents a group -CH (CH 2 SH) -).
- the functionalized support of formula (II) can advantageously be used in the method according to the present invention allowing the attachment, by covalent bond, of at least one nucleic acid M to an SP support, as described above.
- the present invention also relates to a process for preparing the functionalized support of formula (II), in which i is equal to 1, n is equal to 1 and SP represents a glass support, characterized in that it comprises the steps following: silanization of the glass support, grafting, onto said silanized glass support, of a function selected from the group consisting of hydrazine functions, derivatives of hydrazine, hydroxylamine and ⁇ -aminothiol.
- the stage of silanization of the support is preferably carried out with the aid of aminopropyl-trimethoxysilane.
- said grafting of a hydrazine function is carried out using hydrazinoacetic acid
- said grafting of a function derived from hydrazine is carried out using triphosgene and hydrazine
- said grafting of a hydroxylamine function is carried out using aminooxyacetic acid
- said grafting of a ⁇ -aminothiol function is carried out using ⁇ -amino- ⁇ -mercaptopropionic acid.
- the subject of the invention is also a process for controlling the quality of the support of formula (II) as defined above, characterized in that it includes the following steps:
- a fluorescent probe for example rhodamine, derivatized by an ⁇ -oxoaldehyde function
- This method makes it possible to analyze the homogeneity of the functionalization of the support, namely the spatial distribution of the terminal groups -B-NH 2 (cf. formula (II) above).
- the subject of the invention is also a method for quantifying the functionality of the support of formula (II) as defined above, characterized in that it comprises the following steps:
- a fluorescent probe for example rhodamine, derivatized by an ⁇ -oxoaldehyde function
- This method makes it possible to quantify the number of functional sites accessible to the fluorescent probe, on the support of formula (II) above.
- the present invention also relates to a kit for preparing a DNA chip as described above, characterized in that it comprises the following elements: at least one functionalized support of formula (II) according to the present invention, a plurality of oligodeoxynucleotide primers which are modified either in position 3 ', or in position 5', or in position 3 'for a part of said primers and in position 5' for the other part of said primers, by tartaric acid, serine, threonine, their derivatives and the ⁇ -oxoaldehyde group, - reagents and buffers capable of carrying out reactions for elongation and / or amplification of said DNA, and in the case where said oligodeoxynucleotide primers are modified by a group selected from the group consisting of tartaric acid, serine, threonine and their derivatives, reagents capable of carrying out a periodic oxidation reaction.
- the invention further relates to a method for sorting molecules which implements the DNA chip as defined above, as well as the sorted molecules capable of being obtained by this method.
- the invention also comprises other arrangements which will emerge from the description which follows, which refers to examples of implementation of the methods which are the subject of the present invention, as well as to the appended figures, in which:
- FIGS. 1 and 10 show the deprotection of the MMT group carried, respectively, by the oligonucleotide prepared in accordance with Example 1 and by the oligonucleotide prepared in accordance with Example 2,
- FIG. 2 shows, respectively, the coupling of an oligonucleotide with (+) - diacetyl-L-tartaric anhydride, dissuccinimidyl tartrate and trifluoroacetyl-serine, in accordance with Example 1
- - Figures 3, 6 and 8 represent aminolysis reactions of an oligonucleotide immobilized on a solid support, in accordance with Example 1,
- FIGS. 4 and 9 represent periodic oxidation reactions, in accordance with Example 1,
- FIG. 11 represents the coupling of primer 1 with (+) - diacetyl-L-tartaric anhydride
- FIG. 12 represents the aminolysis reaction of the primer 1 immobilized on a solid support, in accordance with Example 2,
- FIG. 13 represents the periodic oxidation reaction of the primer 1, in accordance with Example 2
- FIG. 14 represents the coupling of an oligonucleotide with dissuccinimidyl tartrate, in accordance with Example 3,
- FIG. 15 represents the synthesis of a glass surface functionalized by a hydrazide function, in accordance with Example 5,
- FIG. 16 represents the ligation of a fluorescent probe (rhodamine probe) to a glass surface functionalized with a hydrazide group, for controlling the quality of this surface, in accordance with Example 5
- FIG. 17 represents the synthesis of a rhodamine peptide functionalized with an ⁇ -oxoaldehyde group, in accordance with Example 5, and
- FIG. 18 represents the preparation of a functionalized solid support suitable for the synthesis of oligonucleotides carrying, at their 3 ′ end, an ⁇ -oxoaldehyde function, in accordance with Example 4.
- EXAMPLE 1 Obtaining, by solid phase chemistry, of oligonucleotides modified in position 5 ′ by an ⁇ -oxoaldehyde function.
- the oligonucleotides all have the following sequence: ATCGATCG.
- oligonucleotide of sequence ATCGATCG is synthesized in solid phase, for example on a CPG support (Controled Pore Glass), according to the technique described in "Oligonucleotide Synthesis: a practical approach", edition MJ GAIT, IRL Press, Oxford, 1984, or in “Protocols for oligonucleotides and analogs: synthesis ans properties", edition S. AGRAWAL, Humana Press, Totowa NJ 1993, or by ELLINGTON, A. and POLLARD, JD. in "Current Protocols in Molec lar Biology", 1998, 2.1 1.1-2.11.25, John Wiley & Sons Inc., New York.
- the synthesis follows a classic strategy (5 'hydroxyls protected by dimethoxytrityl groups, chemistry of cyanoethoxyphosphoramidites).
- the bases are protected by acyl groups, which will be labile at the end of the synthesis during aminolysis.
- the 5 'hydroxyl is deprotected and is coupled with an amino spacer arm of formula C ⁇ 2 H 24 -OPO 2 -, protected by a monomethoxytrityl group (MMT).
- MMT monomethoxytrityl group
- the MMT group will be eliminated at the last moment, just before coupling with a tartaric acid derivative, according to the reaction scheme represented in FIG. 1, in which the group P represents the protective group for the nucleotide bases (benzoyl for the bases A and C, isobutyryl for base G).
- oligonucleotide on a support, on the oligonucleotide synthesizer are brought into contact with 3% trichloroacetic acid in dichloromethane, for 5 minutes 30 minutes, by carrying out two intermediate washes with CH CN (acetonitrile) in order to remove the yellow coloration.
- CH CN acetonitrile
- the “supported” oligonucleotide (that is to say the oligonucleotide immobilized on the support) is then washed with CH 3 CN and dried under argon, then with compressed air.
- Two plastic syringes are placed at the two ends of the column in which the oligonucleotide is located on a support.
- 1 ml of NH 4 OH (ammonia) at 32% is placed in the water.
- a mechanical system makes it possible to push the plunger of this syringe in order to deliver 250 ⁇ l of fresh ammonia every 15 minutes.
- the oligonucleotide in solution is thus recovered in the other syringe at the other end.
- This solution is then transferred to a screw eppendorf and placed in an oven at 55 ° C overnight.
- the solution is then cooled and 10 ⁇ l are taken in order to assay the oligonucleotide obtained (22.9 OD in 1000 ⁇ l).
- the ammonia solution is evaporated and the residue is taken up in 400 ⁇ l of water.
- RP-HPL C Analysis and purification by RP-HPL C.
- the rest of the mother solution is purified on the column SP 250/10 Nucleosil 300/5 Cl 8 (room temperature, detection at 254 nm, buffer A: 10 mM TEAA in water, buffer B : CH 3 CN, gradient 5 to 40% B in 20 minutes, flow rate 5.5 ml / min).
- the fractions corresponding to the majority product are isolated.
- the fractions are combined and evaporated on a rotary evaporator, taken up in H 2 O, frozen and lyophilized.
- Quantification analyzes by RP-HPLC and by mass spectrometry by electro-nebulization. The residue is taken up in 1000 ⁇ l of water.
- the 260 nm assay makes it possible to calculate an oligonucleotide quantity of 256.28 ⁇ g.
- Analysis by RP-HPLC 51 ⁇ l of the mother solution is injected onto Cl 8 hypersil 250 x 4.6 mm under the same analysis conditions as above. A 99.14% pure product is obtained.
- Analysis by mass spectrometry by electro-nebulization 5 ⁇ l of the oligonucleotide dissolved in H 2 O / i-PrOH 20% / TEA 1% are injected at a concentration of 10 pmol / ⁇ l.
- Analysis by mass spectrometry by electro-nebulization 20% i-PrOH buffer is continuously infused in H O.
- the reaction was carried out on 181.5 ⁇ g of modified oligonucleotide.
- the final oligonucleotide concentration is 0.65 mM, that of NaIO is 5 mM, ie 7.7 eq of NaIO relative to the oligonucleotide.
- the product is frozen and lyophilized.
- the residue is taken up in 3 ml of water.
- the assay at 260 nm gives 112.6 ⁇ g of oligonucleotide, ie a periodic oxidation yield of 63.77%.
- oligonucleotide on a support are transferred to an empty oligonucleotide synthesis column comprising, at its two ends, two gas-tight syringes.
- 4 ⁇ l (85.86 eq) of 2,6-lutidine dissolved in 80 ⁇ l of THF are introduced into the column by one of the two syringes.
- the oligonucleotide is left in contact with this solution the time to dissolve 6.38 mg (46.4 eq) of dissuccinimidyl tartrate in 80 ⁇ l of THF.
- the latter solution is introduced in turn into the column and the mixture is stirred manually for 5 minutes.
- the oligonucleotide in solution is thus recovered in the other syringe, at the other end. This solution is then transferred to a screw eppendorf and placed in an oven at 55 ° C overnight. The solution is then cooled and 10 ⁇ l are taken in order to assay the oligonucleotide obtained (26.9 OD in 1000 ⁇ l). The ammonia solution is evaporated and the residue is taken up in 400 ⁇ l of water.
- CF 3 -CO-Ser (tBu) -OtBu (2 g, 6.39 mmol) is introduced into a 100 ml flask, then 10 ml of a trifluoroacetic anhydride (TFA) / water mixture (7.5 / 2.5). After three hours, 10 ml of TFA are added. After 2 hours of additional deprotection, the TFA and the water are concentrated under reduced pressure.
- Rf 0.46 (CH 2 Cl 2 / MeOH / AcOH 77.5 / 15 / 7.5); N NMR (300 MHz, DMSO) ⁇ (ppm): 3.8 (m, 2H, CH 2 ⁇ ), 4.2 (m, 2H, CH ⁇ ), 9.5 (solid, 1H, NH).
- the coupling was carried out on batches of 1 ⁇ mol of oligonucleotide.
- the primary amine function of the 5 'aminolink is kept protected by the monomethoxytrityl (MMT) protective group and deprotected at the last moment, just before coupling with trifluoroacetyl-serine, as described above.
- MMT monomethoxytrityl
- Tube A 49.5 ⁇ l of lutidine (85 eq), 46 mg of CF 3 -CO-Ser-OH (46 eq) solubilized in 0.5 ml of DMF
- tube B 120 mg of PyBop (46 eq) dissolved in 0.5 ml of DMF (dimethylformamide).
- the support containing the oligonucleotide is conditioned beforehand for 3 minutes with a solution of 49.5 ⁇ l of lutidine in 1 ml of DMF.
- the tube A is then aspirated with the syringe then the tube B, then the mixture is stirred manually for 5 minutes.
- the resin is washed with DMF (3 times 2 minutes), with DCM (2 times 2 minutes), then is dried with argon.
- the protective groups P of the nucleotides are benzoyl for the bases A and C and isobutyryl for the base G.
- Aminolysis (FIG. 8).
- the resin is placed in the presence of 250 ⁇ l of NH 4 OH 32% for 15 minutes.
- the aminolysis solution is recovered. This operation is repeated 3 times.
- the ammonia solutions obtained as well as 1 ml of solution for rinsing the support with 32% ammonia is transferred to a clean, pyrolized glass tube.
- the tube is closed hermetically and left stirring, at 60 ° C, overnight. The next day, the tube is cooled in an ice bath.
- the oligonucleotide solution is transferred to a hemolysis tube.
- the glass tube is rinsed with 1 ml of water and this aqueous solution is transferred to the same hemolysis tube as above. The solution is then evaporated under reduced pressure.
- the fractions corresponding to the product are combined, frozen and lyophilized.
- ES-MS the product is analyzed under the usual conditions. The observed mass is 2728.0.
- EXAMPLE 2 Another example of obtaining, by solid phase chemistry, oligonucleotides modified in position 5 ′ by an ⁇ -oxoaldehyde function.
- the oligonucleotides have larger sequences than that of the oligonucleotides according to the previous example.
- These oligonucleotides will be called, in what follows, primers 1 and 2 and have the following sequences respectively:
- Primer 1 H 2 NC 6 H ⁇ 2 - spacer arm - GTC CAA GCT CAG CTA ATT
- Primer 2 H 2 NC 6 H 12 - spacer arm - GCA GGA CTC TAG AGG ATC
- the primary amine function of the aminolink in position 5 ′ of the oligonucleotide is kept protected by the protective group MMT and deprotected at the last moment, just before coupling with tartaric anhydride.
- FIG. 10 represents the primer 1, in which the protective groups P of the nucleotides are benzoyl for the bases A and C and isobutyryl for the base G.
- the arm spacer with the following formula: -OPO 2 - (OCH 2 CH 2 ) 6 OPO 2 - (OCH 2 CH 2 ) 6 -OPO 2 - 2) Modification of primers 1 and 2. in position 5 '.
- the primers are each taken up in 1000 ⁇ l of water.
- the assay at 260 nm gives 1605.12 ⁇ g of primer 1 and 1771.44 ⁇ g of primer 2.
- Primer 1 represented below: M expected 6458.48, obtained 6454.5.
- M expected 6458.48 obtained 6454.5.
- the residues are taken up in 1000 ⁇ l of water.
- the 260 nm assay gives the following results: 785.4 ⁇ g of primer 1, 500.3 ⁇ g of primer 2 (for the batch oxidized with 1 mM NaIO 4 ) and 521.4 ⁇ g of primer 2 (for the batch oxidized with 5 mM NaIO 4 ).
- the primers are each taken up in 1000 ⁇ l of water.
- the 260 nm assay gives the following results.
- Primer 1 (1 mM NaIO 4 ): 781.4 ⁇ g of oligonucleotide.
- Primer 2 (1 mM NaIO 4 ): 500.9 ⁇ g of oligonucleotide.
- Primer 2 (5 mM NaIO 4 ): 505.6 ⁇ g of oligonucleotide.
- reaction medium is taken up with 2210 ⁇ l of water and purified on the same ion exchange column using the same elution conditions. The product is frozen and lyophilized.
- a 10 ml syringe is placed above a desalting cartridge (Sep Pak Plus Cl 8 Cartridge).
- 7 ml of 95% CH 3 OH are passed therein in water and then 3 times 7 ml of buffer A.
- the 200 ⁇ l of the oligonucleotide solution are then deposited on the cartridge.
- the salts are eluted with 7 ml of buffer A.
- Argogel resin ® -NH 2 pomrifying be used such as CPG supports (Controlled Pore Glass beads). • Protection of the free hydroxyl function by DMT.
- the resin previously functionalized by 3 is conditioned successive washes of 3 minutes in pyridine.
- 1 g of 4,4'-dimethoxytrityl chloride (3 mmol) dissolved in 5 ml of pyridine is added.
- the excess reagents are removed by filtration and the resin is washed by successive washes with pyridine (5 times 3 minutes), with 3% triethylamine in DCM (3 times 3 minutes) before being vacuum dried.
- the periodic oxidation is then carried out by the following steps.
- the oxidized product is purified by RP-HPLC and lyophilized.
- the majority product is isolated: 213.93 ⁇ g of oligonucleotides (yield of 11%) by quantitative measurement of the OD. Lyophilization in the presence of 1426.9 ⁇ g of mannitol and 0.189 ⁇ l of tri-N-butyl-triphenylphosphine.
- the surface of the glass slides needs to be suitably arranged beforehand.
- the presence of a spacer arm can be useful for moving the probe away from the surface and obtaining optimal hydration, as well as for partially controlling the physicochemical properties of the surface (hydrophilicity, hydrophobia, charge).
- the surface can also be arranged to increase the number of functional sites per unit of surface, for example by the synthesis of dendrimeric structures on glass (BEIER, M. et al., Nucleic Acids Research, 1999, 27, 1970-1977) or by the coupling of polyamines.
- the synthesis strategy envisaged is shown in FIG. 15.
- the same reaction is used as that which will be used to fix the oligonucleotides, that is to say a ligation reaction with a compound functionalized with an ⁇ -oxoaldehyde group (cf. FIG. 16).
- a probe for characterizing the slides with high sensitivity was chosen: it is a fluorescent peptide functionalized by an ⁇ -oxoaldehyde group.
- a rhodamine peptide functionalized by an ⁇ -oxoaldehyde group of sequence (5) -6-carboxytetramethylrhodamine-Lys-Arg-NH- (CH) -NH-CO-CHO was synthesized from the support called IPT (2,3-O -isopropylidene-D-tartrate) described by JS FRUCHART et al. in Tet. Zetters, 1999, 40, 6225-6228 and in the PCT International Application published under the number W ⁇ 00/64843. The synthesis is summarized in FIG. 17.
- the resin is washed with NMP (2 x 2 min), then with DCM (2 x 2 min).
- the side chain protections and the isopropylidene group are deprotected with 5 ml of TFA in the presence of scavengers (375 mg of phenol, 125 mg of ethanedithiol, 250 ⁇ l of thioanisole and 250 ⁇ l of water).
- the resin is then packaged in 5 ml of 33% acetic acid for 2 minutes.
- the peptide is then cleaved from the support by the addition of sodium periodate (0.147 g, 6 eq) diluted in 1 ml of water with stirring for 5 minutes.
- the resin is filtered and then washed with 10 ml of water (3 times 1 minute).
- the cleavage solutions are combined and added to 21 ⁇ l of ethanolamine (3 eq) before being immediately purified on a Cl 8 RP-HPLC Hype ⁇ rep column (15 x 300mm). 8 mg
- Protocol 1 the slides are immersed for 2 hours by ultrasound in a solution of K HPO at 5% in water. The slides are rinsed successively by bathing for 3 minutes in water (2 times) and finally in methanol (1 time). The slides are then dried in a vacuum desiccator.
- Protocol 2 the slides are washed with a tris (hydroxymethyl) - aminomethane-acetate buffer 100 mM pH 5.5 containing 0.1% by mass of Tween 20 for 15 min.
- Pre-cleaned microscope slides with ground edges and frosted margin are immersed in a 10% soda bath in water, first with ultrasound for 10 minutes and then overnight without ultrasound. After having rinsed these slides by three successive baths of 3 minutes in water, they are immersed for 4 hours in a solution of hydrochloric acid at 3.7% in water. Three-minute preliminary rinses are carried out with water (3 times) then with methanol (1 time), before immersing the slides in a bath at 3% aminopropyl-trimethoxysilane in 95% methanol for 30 minutes with ultrasound. The slides are rinsed successively by baths for 3 minutes in methanol (1 time), water (2 times) and finally methanol (1 time).
- the slides functionalized with an isocyanate group are immersed for 2 hours by ultrasound in a solution of Fmoc-NH-NH 2 (22mmol / l) in DMF.
- the slides are then rinsed successively by baths for 3 minutes in DMF (1 time), with water (2 times) and finally with methanol (1 time) before being dried and stored in a vacuum desiccator .
- the slides functionalized by an isocyanate group could be reacted with hydrazine (1% by volume) in DMF. • Protection.
- the process corresponding to the best conditions tested is as follows: the previously obtained slides are immersed in a DMF solution containing piperidine (0.2% by volume) and diazabicyclo-undecene (DBU, 2% by volume) for 30 minutes . The slides are then rinsed successively by baths for 3 minutes in DMF (1 time), with water (2 times) and finally with methanol (1 time) before being dried and stored in a vacuum desiccator .
- Other deprotection systems could consist, for example, of mixtures DMF / piperidine (80/20) or DMF / piperidine / DBU (96/2/2), the contact times with the blades then being respectively 30 minutes or between 2 and 30 minutes. 3) Revelation, quality control of the blades.
- the silanization of the veneer blades is carried out as described by BEIER, M. et al, in Nucleic Acids Research, 1999, 21, 1970-1977 and by BURNS, N. L. et al. in Langmuir, 1995, il, 2768-2776.
- the slides are treated with an aqueous sodium hydroxide solution (10%) overnight, washed with water, with 1% hydrochloric acid in water, again with water and finally with methanol. After sonication for 15 minutes in 95% methanol containing 3% by volume of aminopropyltrimethoxysilane, the slides are washed with methanol, then with water, and dried under a stream of nitrogen. They are heated for 1 minute at 110 ° C. After cooling, they are stored under nitrogen.
- b) Functionalization of the veneer blades • By a hydrazine function.
- Fmoc-hydrazinoacetic acid (Fmoc: 9-fluorenylmethoxycarbonyl) of formula Fmoc-NH-NH-CH -COOH is synthesized from the hydrochloride of ethyl ⁇ -hydrazinoacetate (ALDRICH) by saponification of the ester function in soda followed by protection of the hydrazine function, according to the protocol described by ATHERTON, E. in The Peptides, 1987, 9, part C, Udenfriend S. and Meienhofer J. Eds., Académie Press, San Diego, California.
- ATHERTON ethyl ⁇ -hydrazinoacetate
- the silanized glass slides are brought into contact with Fmoc-hydrazinoacetic acid (100 mM) in the presence of BOP (100 mM) and DIEA (diisopropylethylamine; 200 mM) in dimethylformamide (DMF), for 1 hour.
- BOP 100 mM
- DIEA diisopropylethylamine; 200 mM
- DMF dimethylformamide
- the slides are then washed with DMF, treated with 20% by volume piperidine in DMF for 5 minutes (departure of the Fmoc groups protecting the hydrazine functions), then washed with DMF, with methanol, and dried under nitrogen. • By a hydroxylamine function.
- the silanized veneer blades are treated with Fmoc-aminooxyacetic acid of formula Fmoc-NH-O-CH 2 -COOH (SENN CHEMICALS; 100 mM) in the presence of BOP (100 mM) and DIEA (200 mM) in the DMF, for 1 hour. They are washed with DMF and treated, for 5 minutes, with piperidine at 20% by volume in DMF (departure of the Fmoc groups protecting the hydroxylamine functions). They are then washed with DMF, with methanol and dried under nitrogen.
- Fmoc-aminooxyacetic acid of formula Fmoc-NH-O-CH 2 -COOH
- the silanized veneer blades are treated, in the presence of BOP (100 mM), DIEA (200 mM) and in DMF, for 1 hour, with Fmoc-Cys acid (StBu) -OH (acid of formula Fmoc- NH-CH (CH 2 SStBu) -COOH, corresponding to ⁇ -amino- ⁇ -mercaptopropionic acid whose thiol and amino functions are respectively protected by StBu and Fmoc groups; NOVABIOCHEM, 100 mM). After washing with DMF, they are treated with 20% piperidine in DMF for 5 minutes (departure of the Fmoc groups protecting amino functions).
- EXAMPLE 7 Ligation of nucleic acids functionalized with an ⁇ -oxoaldehyde group to a support functionalized with hydrazide groups, for obtaining DNA or oligonucleotide arrays in accordance with the present invention.
- the ligation of nucleic acids functionalized in position 5 ′ by an ⁇ -oxoaldehyde function on a blade of vene functionalized by a function hydrazide is described below.
- the binding of nucleic acids to the glass slide results in the formation of hydrazone bonds (semicarbazone bonds).
- the efficiency of the ligation on these slides was evaluated by hybridization of complementary oligonucleotides labeled in 5 ′ with a molecule of cyanine-3 (Cy-3).
- the oligonucleotides were deposited on veneer blades using an Affymetrix ® 417 Arrayer robot (Affymetrix Inc., 3380 Central Exwy, Santa Clara, CA 95051) equipped with a sampling head with "needle - and - ring” mechanisms (4 needles).
- the needles have a diameter of 125 ⁇ m and deposit a circular shape of approximately 150-170 ⁇ m in diameter for a volume of approximately 30-50 ⁇ l (volume announced by the supplier). The deposits were spaced 375 ⁇ m from center to center.
- Detection of the fluorescent hybridization probe is obtained using an Affymetrix 8 '418 Array Scanner equipped with 2 laser diodes allowing reading at excitation wavelengths of 532 and 635 nm.
- the fluorescence emitted by the fluorochromes after excitation is detected by a photo-multiplier tube (PMT).
- PMT photo-multiplier tube
- the result is obtained in the form of a 16-bit image file with a resolution of 10 ⁇ m / pixel.
- the computer analysis of the image files and the quantification of the fluorescence intensity was done using the freeware "ScanAlyze" developed by M. EISEN, of Stanford University.
- PBS Phosphate buffered saline
- the oligonucleotides carrying an ⁇ -oxoaldehyde function in the 5 ′ position are as obtained in Example 2 above.
- the oligonucleotides used in this ligation protocol correspond to the following formulas
- Pl- ⁇ -oxo 5'- ⁇ -oxo-GTC CAA GCT CAG CTA ATT-3 ';
- PI -diol 5'-diol-GTC CAA GCT CAG CTA ATT-3 ';
- PI -tartrate 5'-tartrate-GTC CAA GCT CAG CTA ATT-3 '.
- sequences of the complementary oligonucleotides labeled with Cy3 are:
- Complementary P2-Cy3 5'-Cy3-GAT CCT CTA GAG TCC TGC-3 '
- the functionalized oligonucleotides are diluted in water and kept at -20 ° C. until use. The quantity necessary for the deposits is taken from this stock and lyophilized before being resuspended in the deposit solution.
- the veneer blades used are functionalized by a hydrazide group and are as obtained in Example 5 above.
- Different quantities of lyophilized oligonucleotides were resuspended in 20 ⁇ l of solution in order to obtain concentrations of 0.1 mM, 0.05 mM, 0.01 mM and 0.001 mM.
- Different resuspension solutions have been tried in order to obtain the best possible spot appearance.
- the deposits were made at 375 ⁇ m from each other, at a temperature of 20 ° C. and in an atmosphere at 70% ( ⁇ 5%) of relative humidity. After deposition, the slides were incubated in a humidity saturated enclosure (close to 100 relative humidity) at 37 ° C. for 14 to 16 h.
- the slides were then washed in a 0.1% SDS solution for 5 minutes at room temperature in order to remove the oligonucleotides which did not react with the slide.
- This washing step has been optimized in order to eliminate the maximum aspecific adsorption between the oligonucleotide and the vene.
- the slides are dried by centrifugation (5 minutes; 30 xg; 20 ° C) in a vertical position.
- Hybridizations are done in “CMT-Hybridization Chambers” (Corning).
- the deposition zone is prehybridized with 15 ⁇ l of prehybridization buffer (50% formamide, 4x SSC, 0.5% SDS; 2.5x Denhardt) at 50 ° C for 1 h 30 min.
- the prehybridization solution is placed between blade and coverslip.
- the slide is placed in the hybridization chamber which contains 2 reservoirs receiving approximately 15 ⁇ l of prehybridization buffer to saturate the atmosphere inside the chamber with humidity.
- the room is sealed and immersed in a water bath at 50 ° C.
- the chamber is opened, the coverslip is removed and the prehybridization buffer is removed by tilting the slide on absorbent paper.
- 15 ⁇ l of hybridization buffer (50% formamide; 6x SSC; 0.4% SDS; 4x Denhardt; 0.01 mM of additional oligonucleotide) are prepared and incubated 5 min at 95 ° C before being placed on the zone of deposit.
- the slide is covered with the coverslip and placed in the hybridization chamber to be incubated at 50 ° C for 14 to 16 h. This process should be done as quickly as possible to avoid drying out after having removed the coverslip.
- the slide After hybridization, the slide is immersed in 50 ml of 2x SSC in the vertical position in order to take off the coverslip. After detachment, the slide is successively washed in 50 ml of 0.1% SDS, 0.1 x SSC for 5 min; 50 ml of 0.1 l SSC for 5 min; 50 ml of 0.1 x SSC for 5 min. These washes are done in 50 ml Falcon tubes, at room temperature. Agitation is ensured by inverting the tube lx every minute. After the last washing, the slides are rinsed under a jet of sterile water and dried immediately by centrifugation (5 minutes; 30 xg; 20 ° C). • Reading of the slides with the scanner.
- the slides are scanned at 532 nm wavelength (Cy-3), immediately after washing.
- the reading is done at different settings of the laser power and the opening of the PMT tube.
- a standard setting (35% laser power,
- the oligonucleotides PI -tartrate and P2 were engaged in a PCR on the plasmid pFus II comprising a fragment of the S 1 gene from bordetella pertussis: pFus II + SI (the amplified part corresponds to the gene fragment inserted).
- the PCR was performed using AmpliTaq Gold ® from Perkin Elmer and under the conditions recommended by the supplier.
- the amplification cycles are as follows: lx
- the deposition and ligation of these PCR products were carried out under the same conditions as in 3) above.
- the hybridization was carried out as indicated in 4) above using a probe synthesized by unidirectional PCR on the plasmid pFus II + SI and using the oligonucleotide P2 to initiate the synthesis. After hybridization, the slides were washed and analyzed as described in 4) and 5) above.
- Table 2 Fluorescence intensity of oligonucleotides deposited, in a tris-acetate buffer medium pH 5.5 + 450 mM NaCl, on a hydrazide slide and hybrids with complementary sequences labeled with Cy3.
- An aspecific adsorption control is obtained by depositing an oligonucleotide with the same nucleotide sequence as Pl- ⁇ -oxo but whose functionalization is not complete (stop at the diol step) and which therefore does not have the possibility of reacting with the semicarbazid functions of the support.
- This oligonucleotide is deposited at a concentration of 0.1 mM and has an intensity of fluorescence much lower than the Pl- ⁇ -oxo equivalent
- Table 1 allow the estimation of the aspecific fixation intensity as representing only about 4% of the signal (10871 of 0.1 mM Pl- ⁇ -oxo by compared to 441 for 0.1 l mM Pl-d ⁇ ol).
- the t ⁇ s-acetate buffer deposits (Table 2) have the same characteristics as in 3x SSC (Table 1), but with lower fluorescence intensities
- the deposition (in 3x SSC) and hybridization protocols detailed above were repeated several times under the same conditions and identical results were obtained.
- the same concentration range was prepared and deposited using an oligonucleotide of different sequence P2- ⁇ -oxo.
- the deposition and the fixation on a hydrazide slide were carried out under the same conditions as for Pl- ⁇ -oxo
- the hybridization was carried out using an oligonucleotide labeled with a Cy3 at 5 'and complementary to P2. After hybndation, the fluorescence measurements are very comparable to those obtained with Pl- ⁇ -oxo, proving that the results obtained with Pl- ⁇ -oxo are verified with pairs of oligonucleotides of different sequences.
- results presented in this example show that it is possible to fix oligonucleotides functionalized in 5 ′ by an ⁇ -oxoaldehyde function on a hydrazide plate and that the oligonucleotides, once fixed, remain accessible for hybridizations with complementary oligonucleotides. It is also possible to dehybridize, by heating to 95 ° C., the oligonucleotides attached to the slide and to reuse this slide in a new hybridization. As for the oligonucleotide carrying, at its 5 ′ end, a tartrate group, it can be engaged in a PCR reaction then oxidized before being deposited on the hydrazide slide.
- EXAMPLE 8 Deposit of oligonucleotides modified in position 5 ′ by an ⁇ -oxoaldehyde function on glass slides functionalized with hydrazine, hydroxylamnine or ⁇ -aminothiol groups. a) Case of the veneer blades functionalized by hydrazine or hydroxylamine groups.
- the oligonucleotides modified in the 5 ′ position by an ⁇ -oxoaldehyde function are taken up in solution in a phosphate buffer of pH 6.0, then deposited manually or using a robot on the slides of vene obtained in Example 6.
- the deposit is accompanied by the immobilization of the oligonucleotides on the surface by the formation of hydrazone bonds (in the case where the surface carries a hydrazine function) or oximes (in the case where the surface carries a hydroxylamine function).
- the slides are incubated in a humid chamber overnight at 37 ° C. They were washed with water, then undergo a stripping treatment with disodium phosphate (Na 2 HPO 4 ; 2.5 mM) and 0.1% by volume of SDS (sodium salt of the ester of dodecyl sulfate) at 95 ° C and for 30 seconds. After washing with water, the slides are dried under a flow of nitrogen and stored under an inert atmosphere. b) Case of the veneer blades functionalized by ⁇ -aminothiol groups.
- the oligonucleotides modified in position 5 ′ by an ⁇ -oxoaldehyde function are taken up in solution in a phosphate buffer of pH 6.0 containing 1 mM of TCEP (tris- (carboxyethyl) phosphine hydrochloride), then deposited manually or using a robot on the glass slides obtained in Example 6.
- TCEP tris- (carboxyethyl) phosphine hydrochloride
- the protocols described in a) and b) above can also use oligonucleotides modified in position 3 ′ by an ⁇ -oxoaldehyde function, or larger nucleic acids, such as DNA.
- EXAMPLE 9 Ligation between a peptide having a hydrazine function in the N-terminal position and an oligonucleotide having a ⁇ -oxoaldehyde function in the 5 'position.
- This example illustrates the ligation of an oligonucleotide in accordance with the invention to a non-solid support of peptide nature.
- the peptide synthesis was carried out according to the Fmoc / t-Bu strategy on an Applied Biosystems 431 A synthesizer, on 0.25 mmol of MB HA Rink resin
- Amide ® loaded at 0.74 mmol / g.
- the amino acids are activated using a HBTU / HOBt / DIEA mixture (amino acid / HBTU / HOBt / DIEA: 4 eq / 4 eq / 4 eq / 8 eq) in the NMP.
- the side chains are protected as follows: Arg (Pbf), Tyr (t-Bu).
- the resin is divided into 2 lots. On one half (0.125 mmol), the Fmoc is manually deprotected by a piperidine / NMP 20/80 mixture and the triBocGlycineHydrazine is coupled using also HBTU, HOBt and DIEA (4 eq / 4 eq / 4 eq / 8 eq).
- the resin is dried and cleaved for 1 h 30 min with 2.75 ml of a phenol / EDT / thioanisole / H 2 O / TFA mixture (0.3 g / 0.1 ml / 0.2 ml / 0.2 ml / qsp) 4 ml).
- the peptide is precipitated in 200 ml of an Et 2 O / pentane 50/50 mixture. After lyophilization, 42.5 mg of crude peptide are obtained (ie a coupling yield of 45.4%).
- the eppendorf containing the reaction medium, surrounded by parafilm in order to limit evaporation, is placed in a
- the eppendorf is removed from the thermostated bath and 10 ⁇ l of citrate buffer are added 1 hour later.
- the reaction medium is left at room temperature and frozen at -30 ° C after 27 hours. After thawing, 5 ⁇ l of the reaction medium are removed and 55 ⁇ l of water are added thereto.
- SP support can consist of a polyacrylamide tree polymer; in this case, it will be interesting to fix, by covalent bond, of the oligonucleotides on this tree polymer, using the process according to the present invention.
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002393641A CA2393641A1 (fr) | 1999-12-07 | 2000-12-07 | Comprenant un support sur lequel sont fixes des acides nucleiques et leur utilisation comme puce a adn |
JP2001544367A JP2003516159A (ja) | 1999-12-07 | 2000-12-07 | 核酸が固定されている支持体を含むプロダクト、およびdnaチップとしてのそれらの使用 |
EP00988891A EP1235839A2 (fr) | 1999-12-07 | 2000-12-07 | Produits comprenant un support sur lequel sont fixes des acides nucleiques et leur utilisation comme puce a adn |
AU25241/01A AU2524101A (en) | 1999-12-07 | 2000-12-07 | Products comprising a support whereon are fixed nucleic acids and their use as dna chip |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR9915392A FR2801904B1 (fr) | 1999-12-07 | 1999-12-07 | Produits comprenant un support sur lequel sont fixes des acides nucleiques et leur utilisation comme puce a adn |
FR99/15392 | 1999-12-07 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2001042495A2 true WO2001042495A2 (fr) | 2001-06-14 |
WO2001042495A3 WO2001042495A3 (fr) | 2001-12-13 |
Family
ID=9552954
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/FR2000/003427 WO2001042495A2 (fr) | 1999-12-07 | 2000-12-07 | Produits comprenant un support sur lequel sont fixes des acides nucleiques et leur utilisation comme puce a adn |
Country Status (7)
Country | Link |
---|---|
US (1) | US20030162185A1 (fr) |
EP (1) | EP1235839A2 (fr) |
JP (1) | JP2003516159A (fr) |
AU (1) | AU2524101A (fr) |
CA (1) | CA2393641A1 (fr) |
FR (1) | FR2801904B1 (fr) |
WO (1) | WO2001042495A2 (fr) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003099855A1 (fr) * | 2002-05-28 | 2003-12-04 | Sedac Therapeutics | Dispositif de présentation de peptides ou de protéines, son procédé de préparation et ses utilisations. |
JP2005518431A (ja) * | 2002-02-01 | 2005-06-23 | アボット・ラボラトリーズ | 高分子コンジュゲート及びその製造方法 |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2825095B1 (fr) * | 2001-05-28 | 2003-09-26 | Dev Des Antigenes Combinatoire | Dispositif de presentation de polypeptides, utilisable comme "puce" pour la detection miniaturisee de molecules |
WO2005103066A1 (fr) * | 2004-04-09 | 2005-11-03 | Burzynski, Stanislaw, R. | Procede de fixation de sondes moleculaires sur un support solide |
US20060121501A1 (en) * | 2004-10-22 | 2006-06-08 | Stanislaw Burzynski | Method for immobilizing molecular probes to a semiconductor oxide surface |
KR100956447B1 (ko) * | 2008-05-16 | 2010-05-06 | 한국전자통신연구원 | 바이오센서의 기판의 패턴의 제조 방법 및 이를 이용한바이오센서 |
DE102013011304A1 (de) * | 2013-07-02 | 2015-01-22 | Technische Universität Dresden | Verfahren und Anordnung zur Erfassung von Bindungsereignissen von Molekülen |
EP3188733B1 (fr) | 2014-09-03 | 2019-11-06 | ImmunoGen, Inc. | Conjugués comprenant des agents de liaison cellulaire et des agents cytotoxiques |
JP7375998B2 (ja) * | 2018-12-12 | 2023-11-08 | ビージーアイ シェンチェン | バイオチップ、その調製方法及び使用 |
CN112067684A (zh) * | 2019-06-11 | 2020-12-11 | 复旦大学 | 一种基于噻唑烷化学固相富集糖肽并质谱分析的方法 |
CN112552208B (zh) * | 2021-01-25 | 2022-06-10 | 井冈山大学 | 一种用于滴眼液质量检测的荧光分子及其制备和应用 |
-
1999
- 1999-12-07 FR FR9915392A patent/FR2801904B1/fr not_active Expired - Fee Related
-
2000
- 2000-12-07 JP JP2001544367A patent/JP2003516159A/ja not_active Withdrawn
- 2000-12-07 US US10/149,249 patent/US20030162185A1/en not_active Abandoned
- 2000-12-07 WO PCT/FR2000/003427 patent/WO2001042495A2/fr not_active Application Discontinuation
- 2000-12-07 CA CA002393641A patent/CA2393641A1/fr not_active Abandoned
- 2000-12-07 AU AU25241/01A patent/AU2524101A/en not_active Abandoned
- 2000-12-07 EP EP00988891A patent/EP1235839A2/fr not_active Withdrawn
Non-Patent Citations (2)
Title |
---|
FRUCHART J -S ET AL: "A new linker for the synthesis of C-terminal peptide alpha-oxo-aldehydes" TETRAHEDRON LETTERS,NL,ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, vol. 40, no. 34, 20 août 1999 (1999-08-20), pages 6225-6228, XP004174018 ISSN: 0040-4039 * |
M. BEIER, J.D. HOHEISEL: "Versatile derivatisation of solid support media for covalent bonding on DNA-microchips" NUCLEIC ACIDS RESEARCH, vol. 27, no. 9, 1999, pages 1970-1977, XP002145887 cité dans la demande * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005518431A (ja) * | 2002-02-01 | 2005-06-23 | アボット・ラボラトリーズ | 高分子コンジュゲート及びその製造方法 |
WO2003099855A1 (fr) * | 2002-05-28 | 2003-12-04 | Sedac Therapeutics | Dispositif de présentation de peptides ou de protéines, son procédé de préparation et ses utilisations. |
FR2840409A1 (fr) * | 2002-05-28 | 2003-12-05 | Dev Des Antigenes Combinatoire | Dispositif de presentation de peptides ou de proteines, son procede de preparation et ses utilisations |
Also Published As
Publication number | Publication date |
---|---|
JP2003516159A (ja) | 2003-05-13 |
WO2001042495A3 (fr) | 2001-12-13 |
AU2524101A (en) | 2001-06-18 |
EP1235839A2 (fr) | 2002-09-04 |
CA2393641A1 (fr) | 2001-06-14 |
US20030162185A1 (en) | 2003-08-28 |
FR2801904A1 (fr) | 2001-06-08 |
FR2801904B1 (fr) | 2002-02-08 |
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