ITMI20011168A1 - METHOD FOR THE IMMOBILIZATION OF ORGANIC MOLECULES ON A SUPPORT - Google Patents

Description

Description of the industrial invention entitled:

"METHOD FOR THE IMMOBILIZATION OF BIOLOGICAL MOLECULES ON A SUPPORT"

The present invention provides a method for the immobilization of biological molecules, such as nucleic acids, peptides or proteins, on the surface of a solid support of glass or plastic material.

The immobilization of biological molecules on different substrates is of crucial importance in the development of microarrays technology (Nature Genetics Supplement Vol 21, 1999). The latter in recent years have seen a wide diffusion in various sectors of medical-biological research, thanks to their versatility and miniaturization that allows the creation of "high throughput" techniques. The methods and materials used in coating the substrate surfaces used in microarray manufacturing are a decisive factor in the success of these techniques.

In recent years, polymeric coatings based on polyacrylamide or polydimethylacrylamide gels have been developed for regioselective immobilization of the 3 'or 5' end groups of oligonucleotides. The procedure for immobilizing DNA in the polyacrylamide and dimethylacrylamide gel, developed by the Mirzabekov group at the Engelhardt Institute in Moscow (US0981734), involves the formation of functional groups on supporting substrates to facilitate the immobilization of oligonucleotides.

In US05861247 a method for constructing oligonucleotide matrices has been described. The method comprises confining a light-sensitive fluid to the surface, exposing said fluid to a system of light that causes the fluid to coalescence into discrete units and adhere to the surface, and contacting each unit with a group of different oligonucleotide molecules in order to allow the dispersion of the molecules in the units. The procedure for fixing the polyacrylamide gel on the slide is difficult as it requires the silanization of the glass and the photopolymerization of the acrylamide.

According to a different approach, Boles and collaborators at Mosaic Technologies Ine. (US05932711 and US061 80770) have developed the chemistry for the solid phase etching of oligonucleotides based on the synthesis of oligonucleotides bearing acrylamide groups at the 5 'end. The oligonucleotides having polymerizable functions are copolymerized with acrylamide / bisacrylamide and covalently bonded to an organosilane surface on which the acryloyl groups have previously been inserted. This procedure also requires a lot of time and careful control of the operating parameters.

SurModics, Ine. (US06121027) has developed the technology to produce polyfunctional reagents having a polymeric backbone, one or more "bulging" photoreactive regions, and one or more "bulging" bioactive groups. The reactant can be activated to form a bulk material or brought into contact with the surface of previously formed material and activated to form the coating. The "protruding" bioactive groups promote the attachment of specific nucleic acids and other molecules capable of non-covalently binding portions of specific and complementary molecules.

In a recent patent (US05858653) SurModics described a method for producing a polymeric support for the attachment of oligonucleotides and DNA, in which different homopolymers and copolymers, obtained by polymerization by addition or condensation, provide a polymeric skeleton capable of carrying ionic groups , photogroups and thermodynamically reactive groups. The method is based on the activation of latent photoreactive groups to generate active species that are added to other active species on the same or on another molecule in order to generate a three-dimensional network in which the reactive functions are suitably separated from the surface. Although very flexible, this method involves the synthesis of complex copolymers whose composition is difficult to control.

It has now been found, and it is the object of the present invention, a simple and fast method for immobilizing biological molecules, such as peptides, proteins and nucleic acid molecules, in particular DNA, on the surface of glassy or plastic materials commonly used as adhesion substrates for such molecules, such as multiwell plates, beads, test tubes, microscope slides, wafers and silica membranes. Suitable plastic materials are, for example, based on polystyrene, polycarbonate, polyvinyl chloride and polypropylene.

The method exploits the ability of certain acrylamide-based polymers, suitably derivatized, to be adsorbed with considerable affinity on the surface of the materials specified above, thus providing a highly hydrophilic coating matrix to which biological molecules can be covalently bonded. The polymers based on acrylamide, object of the present invention, are obtained by radical copolymerization of a mixture comprising:

a) a monomer selected from the group comprising acrylamide and methacrylamide, preferably mono- or di-substituted nitrogen by linear or branched C1 -C12 alkyl chains, in turn optionally replaced by halogen, preferably fluorine, or by a methoxy or group, in the case of nitrogen mono-substituents, from a hydroxy group; b) an ethylene monomer containing groups capable of reacting covalently with the free amino or thiol groups present in the peptides / proteins or with the amino-modified residues of nucleic acid molecules, these reactive groups being separated from the ethylene unit by means of arms spacers containing aliphatic chains or rings bearing polar groups and / or heteroatoms, in particular linear or cyclic C1-C6 alkyls possibly interrupted by atoms of O, N, S, by carbonyl groups (-CO-), amide (-CONH-), sulfoxides (-SO-), sulfonyl (-S02-), sulfonamides (-S02NH-), carbonyloxy (-COO-).

The content of monomer (b) in the polymerization mixture can vary between 0.1 and 25% w / v, preferably 1-4%, the remainder being made up of monomer (a). In addition, the polymerization mixture may contain a further monomer (c) which differs from monomer (b) by the presence of a group which assumes a positive charge in aqueous or slightly acid solution and which cooperates with monomer (b) in increasing the bond of the biological molecule by combining covalent bond with electrostatic interaction. The monomer content (c) in the polymerization mixture can reach 25% w / v, the 5-10% range being preferred. With the insertion of the monomer (c) it is possible to immobilize the biological molecule in the matrix of the coating polymer by means of a covalent and ionic bond. Among the groups capable of covalently binding biological molecules (monomer (b)), without limiting the invention in any way, active esters of carboxylic acids such as the N-carbonyloxy-imide residue (-COO-N- imide), where the imide is preferably cyclic with 4-5 carbon atoms and optionally substituted, such as succinimide and maleimide, or optionally substituted oxiranes; among the groups capable of establishing electrostatic interactions with the same biological molecules, the amines and quaternary ammonium groups are mentioned. Such groups can already be present in one or more of the monomers of the initial polymerization mixture or they can be introduced after polymerization, as described in Timofeev et al., Nucleic Acid Research, 1996, 24, 16, 3142-3148. In this last work, for example, the preparation of a copolymer N, N-dimethylacrylamide / 2-hydroxyethylacrylate, subsequently treated with methylsulfonic acid chloride to generate a terminal methylsulfonate which represents an excellent leaving group for nucleophilic substitutions, for example with a primary amino group of an aminomodified oligonucleotide.

Preferably N, N-dimethylacrylamide is used as monomer (a), as monomers (b) the allyloxyalkyl (Cl-C4) oxiranes and N-acryloxy [alkyl (Cl-C4)] nsuccinimide (n = 0.1), being more preferred allyloxymethyloxirane and N-acryloxyloxysuccinimide; as monomer (c) Ν, Ν, Ν, -trimethylaminoethylacrylamide is preferably used.

Preferred polymers are obtained starting from the following mixtures of monomers:

Ν, Ν-dimethylacrylamide and N-acryloxyloxysuccinimide ((a) (b));

Ν, Ν-dimethylacrylamide, N-acryloxyloxysuccinimide and Ν, Ν, Νtrimethylaminoethylacrylamide ((a) (b) (c)).

The polymerization reaction can be carried out in non-polar organic solvents, preferably tetrahydrofuran, and is generally catalysed by radical type catalysts such as α, α's-azoisobutyronitrile AIBN.

According to the method of the invention, the polymers described above are adsorbed on the surface of the substrate by placing an aqueous solution of the same in contact with said surface for a time which may vary according to the mixture used and the surface to be treated, but which will generally vary from a few seconds to 30 min or more, in order to form a highly hydrophilic coating in which the reactive functions or groups are accessible to biological molecules. Subsequently, the latter are covalently linked to said reactive groups according to procedures known to the expert in the field (DNA Microarrays A Practical Approach, Mark Schena Ed., Oxford University Press). The aqueous solutions used for the deposition of the polymer coating have a polymer content ranging from 0.1 to 20% w / v, preferably from 0.1. at 1% w / v. In a preferred embodiment, the aqueous solution contains ammonium sulfate at 20% saturation.

The affinity for the substrate is such that the adsorption of the polymers generates a layer that cannot be removed from the substrate surface by common aqueous buffers, even in the presence of additives such as urea, SDS, salts or in high temperature conditions.

According to a preferred embodiment, the polymers are used for coating microarrays of DNA, peptides or proteins, which can be used in hybridization techniques with complementary molecules. Examples of complementary molecules are represented by antigen / antibody, ligand / receptor, enzyme / substrate, protein / protein pairs, preferably nucleic acid molecules that can be used in hybridization techniques in ways well known to those skilled in the art. The invention also includes substrates of plastic or glass material, such as multiwell plates, beads, test tubes, microscope slides, wafers and silica membranes, having surfaces coated with the polymers described herein.

Brief description of the figures

Fig. 1: Fluorescence intensity of the spots as a function of the concentration of the coating matrix of the slides.

A 5 'amino-modified oligonucleotide (Mdw5 by MWG-BIOTECH) in concentration 50 fm / nl (femtomol / nanoliter) was deposited on p-matrix coated slides (DMA-NAS 2%) in concentration 0.2, 0.4, 0.6, 0.8 and 1% in a 20% saturated ammonium sulfate solution. The oligo was then hybridized, following the described protocol, with the complementary probe labeled at 3 'with Cy5 (Mdw4 by MWG-BIOTECH) for 2 hours at 65 ° C. After a series of washes, the slides were read with the mFAr 2000-1 scanner, designed and built by Virtek for Medway, and the image was analyzed using Virtek ChipReader software. Each point represents the average of the fluorescence intensity, in arbitrary units, of 6 spots of the Mdw5 oligo.

Fig. 2: Fluorescence intensity of the spots as a function of the solvent in which the p matrix has been dissolved (DMA-NAS 2%).

A 5 'amino-modified oligonucleotide (Mdw5 by MWG-BIOTECH) in a concentration of 50 fm / nl was deposited on slides coated by the p matrix (DMA-NAS 2%) at 1% dissolved either in tetrahydrofuran (THF) or in water or in a 20% saturated ammonium sulfate solution. The oligo was then hybridized, following the described protocol, with the complementary probe labeled at 3 'with Cy5 (Mdw4 by MWG-BIOTECH) for 2 hours at 65 ° C. After a series of washes, the slides were read with the Mfar 2000-1 scanner, designed and built by Virtek for Medway, and the image was analyzed using the 1D Image Analysis software by Kodak Digital Science. Each bar represents the average of the fluorescence intensity of 6 spots.

Fig. 3: Average fluorescence intensity of spots as a function of the oligonucleotide concentration.

Each point represents the average of the fluorescence intensity, in arbitrary units, of 6 spots of a 5 'amino-modified oligonucleotide (Mdw5) (by MWG-BIOTECH) at concentrations of 1, 5, 25 and 50 frn / nl. The amino-modified oligonucleotide, deposited on a glass support coated with the matrix p (DMA-NAS 2%) at 1% concentration, was hybridized, following the described product, with the complementary probe (Mdw4) labeled at 3 'cpm Cy5 (by MWG-BIOTECH) for 2 hours at 65 ° C. After a series of washes, the slide was read with the Mfar 2000-1 scanner, designed and built by Virtek for Medway, and the image was analyzed using the 1D Image Analysis software by Kodak Digital Science.

Fig. 4: Oligo-oligo hybridization experiment.

A 5 'amino-modified oligonucleotide (Mdw5 by MWG-BIOTECH) in concentrations 10 fm / nl (left column), 25 fm / nl (middle column) and 50 fm / nl (right column) was deposited on a slide coated with 1% p (DMA-NAS 2%) matrix dissolved in a 20% saturated ammonium sulphate solution. The oligo was then hybridized, following the described protocol, with the complementary probe labeled at 3 'with Cy5 (Mdw4 by MWG-BIOTECH) for 2 hours at 65 ° C. After a series of washes, the slide was read with the Mfar 2000-1 scanner, designed and built by Virtek for Medway.

Fig. 5: Oligo-cDNA hybridization experiment.

A 5 'amino-modified oligonucleotide (Mdw 14 by MWG-BIOTECH), corresponding to a fragment of the neomycin gene derived from the pEGFP-Nl plasmid, in concentrations 3.125, 6.25, 12.5, 25 and 50 fm / nl (from left to right ) was deposited on a slide coated with 1% p (DMA-NAS 2%) matrix dissolved in a 20% saturated ammonium sulphate solution. The oligo was then hybridized for 2 hours at 42 ° C, following the described protocol, with the complementary cDNA fragment of the neomycin gene, labeled at 3 'with a Cy5. After a series of washes, the slide was read with the mFAR 2000-1 scanner, designed and built by Virtek for Medway.

The following examples illustrate the invention in greater detail.

EXAMPLES

Example 1

Synthesis of N-acryloxyloxysuccinimide

Acryloyl chloride (0.99 g, 11.0 mmol) cooled to 0 ° C, was added dropwise to a solution of N-hydroxysuccinimide (NAS) (1.15 g, 10.0 mmol) and triethylamine (1.53 ml) in chloroform (15 ml), under mechanical stirring over a period of 30 minutes. After further stirring for 20 minutes at 0 ° C, the solution was washed with ice-cold water (18 ml x 2 times), dried over Na2SO4 and filtered. 2,5-di-tert-butylhydroquinone (0.5mg) (polymerization inhibitor) was added to the chloroform solution, which was concentrated to a volume of 3ml using a rotary evaporator and filtered.

Ethyl acetate (3 ml) and hexane (2 ml) were added slowly with stirring to the chloroform solution, and the mixture was left at 0 ° C for several hours. A precipitate is thus formed, a colorless solid, which is separated by filtration and washed with an ice-cold solution of ethyl acetate / n-hexane (4/1) and then washed with n-hexane.

13C-NMR (DMSO), δ (ppm): 150 (carbonyl), 137 (CH2 =), 122 - (CH =), 24.8 (-CH2-)

Example 2

Synthesis of poly (N, N-dimethylacrylamide-co- N-acryloxyloxysuccinimide) [DMA98-co-NAS2].

In a 25 ml flask equipped with a condenser, magnetic stirrer and nitrogen connection, N, N-dimethylacrylamide (600 mg, 6.15 mmol) and N-acryloylsuccinimide (20.7 mg, 0.12 mmol) were dissolved in 6 ml of anhydrous tetrahydro furan (THF). The solution was degassed by alternating nitrogen delivery with vacuum for a period of 30 minutes. Two mg of α, α'-azoisobutyronitrile (AIBN) was added to the solution which was then heated to 50 ° C and left at this temperature under slightly positive hydrogen pressure for 24 hours. Upon completion of the polymerization, the solution was evaporated using a rotary evaporator, the white solid was dissolved in chloroform and precipitated by adding petroleum gasoline. The supernatant was discarded and the entire procedure repeated twice. The polymer was dried under vacuum for 24 hours at room temperature and stored at 4 ° C.

<13> C-NMR (DMSO), δ (ppm): 174.6 (carbonyl of the skeleton), 166 (carbonyls of succinimide), 40 - 30 (carbonyls of methylene). The degree of incorporation of succinimide, determined by the ratio of skeletal carbonyl integrals to succinimide, is 1.5%.

Example 3

Synthesis of [DMA90-coNAS10].

The synthetic path is the same as described above with the only difference being the ratio between DMA (600 mg, 6.15 mmol) and NAS (103.4 mg, 0.61 mmol). 13C-NMR (DMSO), δ (ppm): 174.6 (carbonyl of the skeleton), 166 (carbonyls of succinimide), 40 - 30 (carbonyls of methylene). The degree of incorporation of succinimide, determined by the ratio of the integrals of the skeleton carbonyl and succinimide, is equal to 7%.

Example 4

The synthetic path is the same as described above with the only difference being that a mixture of three monomers is polymerized: A, A-dimethylacrylamide (600 mg, 6.15 mmol), A-acryloylsuccinimide (20.7 mg, 0 , 12 mmol) and Ν, Ν, Ν-trimethiaminoethylacrylamide (47 mg (0.3 mmol) in 6 ml of anhydrous tetrahydrofuran (THF).

Example 5

Assay for the active ester content of [DMAn-co-NASm], with n = 98, m = 2; and n = 90, m = 10, in aqueous solution.

It has been observed that N-hydroxysuccinimide has no absorption in UV at 260 nm; however, in alkaline conditions, an absorption peak was detected at this wavelength due to the presence of the anionic species 1, λ max = 260 nm, ε - 9700 M <-1> cm <-1>.

Therefore the appearance of 1 following alkaline hydrolysis can be used to evaluate the quantity of NAS incorporated in the polymer and freely accessible to hydrolysis. The appearance of 1 was followed spectrophotometrically at 260 nm at 25 ° C. Upon completion of the reaction and after zeroing of the absorbance increase, the active ester concentration was calculated from the extinction coefficient of 1.

[DMA98-co-NAS2] and [DMA90-co-NAS10] contain respectively 90 and 400 pine nuts of ester groups of N-hydroxysuccinimide / g of polymer indicating that the accessible NAS groups are approximately 1% and 4%.

Example 6

Assay of the active ester content of [DMAn-co-NASm], with n = 98, m = 2; and n = 90, m = 10, incorporated on the surface of the test piece.

One tube (6 cm high x 0.8 cm wide) was coated with a polymer solution bearing NAS groups. The determination of the NAS groups accessible to hydrolysis after adsorption of the polymer on the surface was carried out by recording the change in absorbance at 260 nm of the ammonium solution used to hydrolyze the NAS groups on the inner surface of the tube. Again, the increase in UV absorption at 260 nm was determined by the production of 1 following hydrolysis.

For [DMA98-co-NAS2], the number of NAS / mm2 of assets was 29.0 pmol / mm2.

Example 7

Coating of glass plates

Coating of glass plates requires two steps, a) surface pretreatment and b) polymer adsorption. In the first step the slides are washed with 1 M NaOH for 30 minutes, with 1 M HCl for 30 minutes, with water and dried. In the second step, the pretreated slides were immersed for 30 minutes in a polymer solution from 0.1 to 1% w / v dissolved in an aqueous solution of ammonium sulphate at 20% saturation. The slides are then washed thoroughly with water and dried in an oven at 60 ° C. The effect of the polymer concentration on the fluorescence intensity after hybridization was examined. Figure 1 shows the fluorescence signal of hybridized oligonucleotides as a function of the polymer concentration used during the adsorption step. The results clearly indicate that a polymer concentration of 1% w / v provides the best results. The addition of ammonium sulfate to the polymer solution also increases the fluorescence signal while the dissolution of the polymer in organic solvent drastically reduces the amount of polymer adsorbed on the surface. Figure 2 summarizes the results in terms of the fluorescence signal obtained with the polymer dissolved in different media.

Example 8

Immobilization of probes

DNA Deposition: Synthetic 3 'aminomodified oligonucleotides and PCR products, previously desalted, were dissolved in 150 mM sodium phosphate buffer at pH 8.5. Stock solutions of 100 nM / mL or 0.1 - 0.5 mg / mL were used for oligonucleotides and PCR, respectively. DNA solutions were diluted to 25, 10 and 5 nM / mL and 1 n spots were deposited on the coated slides to prepare microarrays. Figure 3 shows the fluorescence intensity of the hybrids as a function of the concentration of oligonucleotides located on the surface.

The printed slides were placed in an uncovered cassette which was placed in a sealed chamber saturated with NaCl and allowed to incubate at room temperature. Overnight incubation gives the best results, the minimum incubation period being 4 hours.

Hybridization protocol: the residual reactive groups were blocked by immersion of the printed slides in a 50 mM solution of ethanolamine in 0.1 M Tris, pH 9.0, containing 0.1% sodium dodecyl sulfate (SDS) at 50 ° C for 15 minutes. After removing the block solution, the slides were washed twice with water and shaken for 15 - 60 minutes with 4X SSC / 0.1% SDS buffer, prewarmed to 50 ° C. After a brief washing with water, the slides were treated differently depending on the nature of the probes. In the case of oligonucleotide arrays the slides were placed in the housing and centrifuged at 800 rpm for 3 minutes. In the case of double-stranded DNA arrays, the slides were left in boiling water for 2 minutes, washed twice with water and centrifuged at 800 rpm for 3 minutes. Subsequently, the target molecules (2.5 μΐ per cm <2>), were dissolved in a suitable hybridization buffer, heated in boiling water for 2 minutes, cooled and immediately applied to the microarrays prepared as described above. The slides, placed in hybridization chambers, were transferred to a humidified incubator at a suitable temperature for 4 - 16 hours.

Wash and Scan: Slides were washed twice with 2S SSC / 0.1% SDS at hybridization temperature for 5 minutes. Two washing steps were then carried out with 0.2X SSC and 0.1X SSC. The slides were dried and scanned. Figure 4 shows the results of a hybridization experiment between surface-deposited oligonucleotides and Cy5-labeled oligonucleotides and compares the results with those obtained with commercial slides under identical conditions. Figure 5 shows the results of a hybridization experiment between surface-deposited oligonucleotides and Cy5-labeled PCR products.

Claims (13)

CLAIMS 1. Polymer obtainable by copolymerization of a mixture comprising: a) a monomer selected from the group comprising acrylamide and methacrylamide, unsubstituted or mono- or di-substituted for nitrogen by linear or branched C1 -Cl 2 alkyl chains, in turn optionally replaced by halogen or by a methoxy group or, in case of nitrogen monosubstitutes, from a hydroxy group; b) an ethylene monomer containing groups capable of reacting covalently with the free amino or thiol groups present in the peptides / proteins or with the amino-modified residues of nucleic acid molecules, these reactive groups being separated from the ethylene unit by means of arms hydrophilic spacers containing aliphatic chains or rings bearing functional groups or atoms capable of giving the monomer solubility in water, preferably linear or cyclic C1-C6 alkyls possibly interrupted by atoms of O, N, S, by carbonyl groups (-CO-), amide (-CONH-), sulfoxides (-SO-), sulfonyl (-S02-), sulfonamides (-S02NH-), carbonyloxy (-COO-). 2. Polymer according to claim 1, wherein the groups capable of reacting covalently with the free amino or thiol groups present in the peptides / proteins or with the amino-modified residues of nucleic acid molecules are selected from 4-cyclic N-carbonyloxyimides -5 carbon and oxirane atoms. 3. Polymer according to claims 1-2, further comprising monomer units (c) which differ from (b) by replacing the group covalently binding the biological molecules, with a group which assumes a positive charge in an aqueous environment. 4. Polymer according to claims 1-3, wherein the monomer (a) is N, N-dimethylacrylamide, the monomer (b) is selected from allyloxymethyloxirane and N-acryloxyloxysuccinimide, the monomer (c) is Ν, Ν, Ν- trimethylaminoethylacrylamide. 5. Polymer according to claims 1-3, obtained starting from the following mixtures of monomers: Ν, Ν-dimethylacrylamide and N-acryloxyloxysuccinimide ((a) (b)); Ν, Ν-dimethylacrylamide, N-acryloxyloxysuccinimide and Ν, Ν, Ν-trimethylaminoethylacrylamide ((a) (b) (c)). 6. Method for coating plastic or glass surfaces, which comprises the preparation of an aqueous solution of a polymer according to the previous claims and contacting the same with the surface to be treated for a time sufficient for the polymer to be adsorbed. 7. Method according to claim 6, wherein the aqueous solution has a polymer content ranging from 0.1 to 20%. 8. Method according to claim 7, wherein said aqueous solution contains ammonium sulfate at 20% saturation. 9. Method for the immobilization of biological molecules on the surface of a support of plastic or glassy material which comprises the coating of said surface according to claims 6-8 and the subsequent bonding of the biological molecules by reaction of the free amino or thiol groups therein present with the reactive groups present in the polymer according to claims 1-5. Method according to claim 9, wherein said biological molecules are peptides, proteins or DNA. 1 1. Support of plastic or glassy material for biological molecules having at least one surface coated with a polymer according to claims 1-5. 12. Support according to claim 11, selected from multiwell plates, beads, test tubes, microscope slides, wafers and silica membranes. 13. Use of a support according to claims 11-12 for the immobilization of biological molecules.