WO2005103066A1 - Method for attaching molecular probes to a solid support - Google Patents

Abstract

Disclosed is a novel method for attaching aldehyde, ketone or carboxyamide derivatives of molecular probes to hydrazide or hydrazine treated solid supports under alkaline conditions. In contrast to chemistries commonly used to attach biologically relevant molecules to a solid support (e.g., thiol or primary amine conjugation chemistry), the hydrazone bonds disclosed here exhibit improved chemical properties (e.g., improved stability in water and resistance to changes in pH and temperature).

Description

METHOD FOR ATTACHING MOLECULAR PROBES TO A SOLID SUPPORT CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This PCT application claims priority to Provisional Application Serial No. 60/560,896, filed April 9, 2004, the entire contents of which are incorporated herein by reference. FIELD OF THE INVENTION

[0002] The present invention relates to the chemistry of attachment of molecules to solid surfaces. More particularly, a method for attaching aldehyde, ketone, carboxyamide derivatives of nucleotides, proteins, peptides, lipids, xenobiotics, glycoprotein and their ligands to hydrazide or hydrazine treated solid-supports is disclosed. DESCRIPTION OFTHE RELATEDART

[0003] A wide array of methods have been developed to detect interactions between biological molecules- (e.g., nucleotide-nucleotide, nucleotide-protein, nucleotide-lipid, nucleotide- xenobiotic, protein-protein, protein-lipid, protein-xenobiotic, lipid-lipid, lipid-xenobiotic, glycoprotein-binding factors and their ligands). Some such methods detect interactions between molecular probes attached to solid supports and target molecules in a complex solution. Some of these methods involve derivitized molecular probes in ways that incorporate with free amines or carbonyl groups, depositing (spotting) them on solid supports that have been derivitized to contain complementary free carboxyl or amine groups. For example, U.S. Patent No. 6,339,147 by Lukhtanov et al. describes oligonucleotides (ODNs) that are coupled to a solid support in improved yield resulting in a high density of coupled oligonucleotide per surface unit of the support, through a Schiff base type bond formed between an NH₂ group attached either to the solid support or to the ODN and an aromatic aldehyde attached to the other of the solid support and the ODN. The preferred solid support-ODN conjugate is formed between semicarbazide groups attached to a glass surface and an aromatic aldehyde attached at either 3', or 5' end of an ODN or to an intermediate nucleotide of the ODN.

[0004] Typically, the solid supports are in the form of nitrocellulose, nylon or PDVF membranes, ceramic chips, magnetic beads, glass slides or the wells of polystyrene microtiter plates. A negative feature of deposition methods is that reactive groups along the length of molecular probe react with multiple complimentary groups at surface of the solid support. This results in the probe lying flat on the surface of the support. The juxtaposition of the probe and the support surface reduces the probability of interactions between probes and targets. [0005] Various methods for controlling the alignment of molecules, for example, immobilizing the bound molecules in an upright position on a support surface have been proposed. For example, Pavilckova et al., Biotechniques, 34(1), 124-130 (2003) describes a method for detecting recombinant antibody-antigen interaction in a microarray format using an oriented streptavidin monolayer to provide an interface with well-defined surface architecture that reduces nonspecific binding interactions.

[0006] Kumar et al., Nucleic Acids Research 28(14) (2000) (electronically available at nar.oupjournals.org/cgi/content/full/28/14/e71), describes a method for simultaneous deposition and covalent cross-linking of oligonucleotide or PCR products onto unmodified glass by covalently conjugating an active silyl moiety onto oligonucleotides or cDNA in solution and immobilizing the modified biomolecule onto glass.

[0007] U.S. Patent No. 5,663,242, by Ghosh et al. describes methods for covalent attachment of oligonucleotides to solid supports such that substantially all of the oligonucleotides are attached via their 5'-ends. Thiol-oligonucleotides are attached to bromoacetyl-derivatized polyacrylamide supports, or conversely, bromoacetyl-oligonucleotides are immobilized on thiol-polyacrylamide supports. Ghosh et al. also describe bromoacetyl-derivatized oligonucleotides, and polyacrylamide supports with linked oligonucleotides produced by coupling bromoacetyl- derivatized oligonucleotides with thiol-derivatized polyacrylamide solid supports or by coupling thiol-derivatized oligonucleotides with bromoacetyl-derivatized polyacrylamide supports as well as methods for capture of nucleic acids by oligonucleotides attached to polyacrylamide solid supports, either by direct capture or in sandwich hybridization formats.

[0008] U.S. Patent No. 6,313,284 by Kwiatkowski et al. describes a method of preparing an immobilized oligonucleotide having a free 3 '-end by: i) preparing an oligonucleotide attached in a first position to a solid support via its 3'-end and having a free 5'-end; ii) binding the oligonucleotide in a second position remote from the 3'-end to the solid support; and iii) selectively releasing the 3 '-end of the oligonucleotide from the solid support to obtain the oligonucleotide attached to the support in said second position in a reversed orientation with a free 3 '-end. [0009] Kremsky describes a general method for immobilization of DNA through its 5' - end. Synthetic oligonucleotides, modified at their 5' - ends with an aldehyde or carboxylic acid, are reacted with sodium cyano-borohydride (for aldehydes) or a carbodiimide (for carboxylic acids) and then attached to latex microspheres containing hydrazide residues. The small particles - with diameters of less than 1 micrometer - generally exist as colloidal suspensions. SoluLinK (San Diego, CA) offers bioconjugation kits wherein aldehyde modified beads can be conjugated to hydrazinonicotinamide modified biomolecules. The use of this system however is somewhat cumbersome.

[0010] By the present invention, the end-on attachment of molecular probes to a solid support - that do not exist as a colloidal suspension - is now realized. Nucleic acid probes can be attached end-on using modified groups (3' and/or 5') e.g.: aldehyde., carboxyamide. Surprisingly, the entire attachment can be performed at alkaline pH. Contrary to prior art, hybridization to complimentary target nucleic acids also occur under alkaline conditions. Unexpectedly, zeptomole levels of target nucleic acid can now be detected. This robust method of probe attachment allows the benefits of solution hybridization combined with the capability of ELIS A formats. Additionally, attached probe is not washed away (contrast with colloidal suspension of Kremsky). Custom probes will allow detection of any nucleic acid target (eg. infectious disease, GMO, etc.) - at levels previously unattainable. SUMMARY OF INVENTION

[0011] An aspect of the present invention is a method for attaching or conjugating end-region aldehyde, ketone or carboxyamide derivitized molecular probes with hydrazide- or hydrazine- derivitized solid supports, so that a stable bond is formed between the probe and the solid support. Typically, hydrazone bonds are formed. Molecular probe materials capable of being derivitized include nucleotides, oligonuceotides, proteins, peptides, lipids, xenobiotics, proteoglycans, heparin, biotin or other biological molecules of interest. Any solid support capable of being derivitized with hydrazine or hydrazide can be used in accordance with the invention.

[0012] The present invention provides a method of attaching a molecule having a functional group selected from the group consisting of aldehyde, ketone, and carboxyamide to a solid support. The method involves contacting under alkaline conditions the molecule with a solid support comprising a surface having attached to the surface moieties selected from the group consisting of hydrazine, hydrazide and mixtures thereof. Upon contact, the molecule will covalently bond to the solid support surface via the hydrazine or hydrazide moieties to form a stable hydrazone bond. DESCRIPTION OF THE FIGURES

[0013] Figure 1 is a graph showing the chromogenic detection (after 40 minute development) of a biotinylated DNA oligonucleotide directly attached to hydrazide microtiter plate.

[0014] Figure 2 is a schematic showing hydrazone bond with biotin, streptavidin-alk-phos conjugation and reaction with biotin, chromogenic detection of alk-phos.

[0015] Figure 3 is a schematic showing the chemical structure of biotin and depicting the site of hydrazone bond with a hydrazide microtiter plate.

[0016] Figure 4 is a graph showing the time course of chromogenic development of labelled

DNA, indicating kinetics of assay at 10 and 20 minutes.

[0017] Figure 5 is a schematic showing a of an assay having a hydrazone bond of a 5' aldehyde labeled probe and a hydrazide microtiter plate, subsequent hybridization with 5'-biotinylated target DNA, colorimetric detection via streptavidin-alk-phos incubation, and substrate development.

[0018] Figure 6 is a graph showing the 40 minute read of chromogenic development of hybridized DNA. DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Disclosed herein is an attachment chemistry that conjugates end-region aldehyde, ketone or carboxyamide-derivitized molecular probes to hydrazine or hydrazide-treated solid support structures. The chemical structures of reactive aldehyde, ketone or carboxyamide groups are generally added to the end-regions of the desired molecular probe, as shown below. [0020] Examples of molecular probes that can be derivitized to contain aldehyde, ketone, or carboxyamide reactive groups include, but are not limited to, oligonucleotides, proteins, peptides, lipids, xenobiotics, glycoprotein and their ligands. A number of biologically relevant molecules that are derivitized to contain aldehyde, ketone, or carboxyamide reactive groups can be commercially obtained. For example, 5' -derivitized oligonucleotides are available from Solu-Link (San Diego, CA). Alternatively, probes can be derivatized by methods known to those skilled in the art. A probe derivitized to contain an aldehyde can be represented schematically as: O Probe -CH

[0021] Hydrazine and hydrazide treated solid supports are commercially available (e.g. from Sigma, Chemical, St Louis, MO or Corning Inc. Corning, NY). Essentially any solid support can be treated with hydrazine providing it has a ligand capable of attachment to hydrazine (via hydrazide or hydrazone linkage). Examples of such supports include, but are not limited to, hydrazine treated polyacrylamide, polystyrene, polysulfone, PVDF, glass (as well as glass fiber), polypropylene, Sulfhydryl, Poly-lysine, Protein A, Protein G, Collagen, Streptavidin. Other types of solid supports include microtiter plates, beads, chromatography columns, slides, dipsticks, and membranes. According to a preferred embodiment, the hydrazine-modified support is a CarboBIND™ well plate, available from Corning. A hydrazine-modified solid support can be represented schematically as:

H₂NHNH₂C- Support

[0022] The term "hydrazine-treated" support as used herein is intended to include any support treated with unsubstituted hydrazine (NH₂NH ), one or more substituted hydrazines, and/or mixtures of unsubstituted hydrazine and one or more substituted hydrazines, so long as there is provided on the support a -NHNH₂ moiety that is available for reaction with the derivitized molecular probe. For example, a hydrazide treated solid support containing an exposed acyl group can be represented: O

H NHNC- Support [0023] According to the present invention, probes are bound to the hydrazine-treated support via hydrazone or acyl-hydrazone bonds. The hydrazone bond is particularly stable because it is a double bond. Additionally, hydrazone bonds are stable with respect to shifts in pH and temperature. Hydrazone bonds are superior to other bonds commonly used to immobilize molecules on surfaces (e.g. thiol, Schiff, hydrazine bonds).

Support I — HN=CH — | Probe | Hydrazone bond

[0024] Contacting the derivitized aldehyde, ketone or carboxyamide molecular probe with a hydrazine or hydrazide treated solid support forms the hydrazone or acyl-hydrazone bonds. • While acid conditions can be used, the reaction is preferably performed in alkaline conditions at r. a pH of about 7.5 to 12, with a pH of about 8 to 10 being more preferred and with a pH of about 8.5 to 9.5 being most preferred. The reaction may conveniently be performed at temperatures of about 20 to 40 degrees °C, with room temperature preferred.

[0025] As described above, any molecular probe containing an aldehyde, ketone, or carboxyamide can be immobilized on a hydrazine or hydrazide treated support, thereby providing an architecture for a variety of applications. For example, immobilized oligonucleotides are used in procedures that utilize arrays of oligonucleotides, such as sequencing by hybridization and array-based analysis of gene expression. In the present invention, the attachment of oligonucleotides to a hydrazide modified surface under alkaline conditions helps prevent self hybridization. In sequencing by hybridization, an ordered array of oligonucleotides of different known sequences is used as a platform for hybridization to one or more test polynucleotides, nucleic acids or nucleic acid populations. Determination of the oligonucleotides that are hybridized and alignment of their known sequences allows reconstruction of the sequence of the test polynucleotide. Alternatively, oligonucleotides containing the wild-type sequence and all possible mutant sequences for a given region of a gene of interest can be placed on an array. Exposure of the array to DNA or RNA from a subject or biological specimen, under hybridization conditions, allows determination of wild-type or mutant status for the gene of interest. This is described, without using the present invention, in the prior art, for example in U.S. Pat. Nos. 5,492,806, 5,525,464, and 5,556,752, all of which are incorporated herein by reference. Both of the foregoing techniques require discrimination between related sequences, especially at the single-nucleotide level; hence, the simplicity, reproducibility of solid support attachment oligonucleotides of the invention provides improvements in these techniques.

[0026] An additional application of the present invention to array technology is examination of patterns of gene expression in a particular cell or tissue. In this situation oligonucleotides or polynucleotides corresponding to different genes are arrayed on a surface, and a nucleic acid sample from a particular cell or tissue type, for example, is incubated with the array under hybridization conditions. Detection of the sites on the array at which hybridization occurs allows one to determine which oligonucleotides have hybridized, and hence which genes are active in the particular cell or tissue from which the sample was derived.

[0027] Array methods can also be used for identification of mutations, where wild-type and mutant sequences are placed in an ordered array on a surface. Hybridization of a polynucleotide sample to the array under stringent conditions, and determination of which oligonucleotides in the array hybridize to the polynucleotide, allows determination of whether the polynucleotide possesses the wild-type or the mutant sequence. Since many mutant sequences of clinically- relevant genes differ from their wild-type counterpart at only one or a few nucleotide positions, the enhanced discriminatory powers of the modified oligonucleotides of the invention provides improvements in mutation detection. Array methods can also be used in any diagnostic procedure where nucleic acid hybridization is feasible in combination with an appropriate detection system. The nucleic acids include DNA, RNA and sequences amplified by methods known in the art.

[0028] Alternatively, a peptide, lipid, or xenobiotic probe can be used. For example, a xenobiotic probe can be used to study the interactions between drugs and biological molecules. [0029] Although the present invention is explained in greater detail above, variations and additions to the various embodiments suggested herein would be apparent to those skilled in the art in light of the instant disclosure which does not depart from the instant invention. Thus, the above description is intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all variations, permutations and combinations thereof. EXAMPLES Example 1 : Carboxyamide-Derivitized Molecular Probe:

[0030] various concentrations of a biotinylated "Poly-A" oligonucleotide (12-mer) DNA probes (100 ul / well) were attached to hydrazine treated 96 well microtiter plates (from Corning Life Sciences, Corning, NY) using an overnight incubation (pH 9.0; 23°C). The wells of the microtiter plate were washed twice with PBS. The wells were then blocked (overnight at room temperature) with salmon sperm DNA (100 ul at 100 ug /ml). Residual salmon sperm was aspirated and the wells were washed twice with PBS.

[0031] Probe attachment was confirmed by binding alkaline phosphatase-streptavidin (AP) conjugate to the biotin moiety of the attached probe. The incubation conditions for binding were 50 ul; 5ug/ml AP; 15 min at room temperature. Unbound AP was removed with PBS rinses (2X). The presence of attached probe was detected using patented (US#5,354,658) reagents for detection of alkaline phosphatase from Enzyme Substrates (Stafford, TX): alkaline phosphatase converts the tetrazolium salt - present in the detection reagent - to a coloredformazan - in direct proportion to the amount of enzyme (AP-SA) conjugated to the biotinylated DNA The optical density (630 am) after 40-minute room temperature indicates the quantity of attached probe. As seen in Fig. 1, under these conditions coupling of the probe to the support can be detected when the probe is at zeptomole (1.0 X 10^"21) concentrations. The design of the assay is depicted in Fig. 2. Fig. 3 provides a more detailed view of the hydrazone linkage of biotin to the surface of the plate.

Example 2: Aldehyde-Derivitized Molecular Probe:

[0032] An oligonucleotide probe, derivitized on its 5' end with an aldehyde group, was attached to hydrazine-treated microtiter plates and used to detect target oligonucleotides with a complementary sequence. A 5' aldehyde-derivitized oligonucleotidQ ("Poly-T" 20-mer) from Solu-Link (San Diego, CA) was coupled to hydrazine treated 96 well microtiter plates (Corning Inc., Corning NY) by incubating 100 ul of 1.0 nM probe per well at a pH between 8 and 8.5, at room temperature overnight. The wells microtiter plate were washed free of unreacted probe with PBS. The wells were then blocked (overnight at room temperature) with salmon sperm DNA (100 ul at 100 ug /ml). The residual unbound salmon sperm was aspirated and the wells washed 2X with PBS. A Tris (0.1M, pH 9.0) solution containing various concentrations of a biotinylated DNA target ("Poly A"; 12-mer) was then added to each well and allowed to hybridize with the attached "Poly T" probe overnight at room temperature. Unbound target was rinsed away with PBS. The target was detected by binding alkaline phosphatase-streptavidin (AP) conjugate to the biotin moiety target by incubating 50ul; 5ug/ml AP; 15 min at room of the temperature. The presence of target DNA was detected using reagents from Enzyme Substrates (Stafford, TX). The optical density (630 nm) at various times of chromogenic development, at room temperature, indicates the quantity of target. Fig. 4 shows that under these conditions the detection of the target is dependent on the duration of the chromogenic reaction. DNA can be 91 detected when the target is at zeptomole (1.0 X 10^" ) concentrations.

[0033] The attachment of the probe to the microtiter plate was confirmed by the addition of a solution containing a target DNA molecule (derivitized in a way that allow its detection). Following the hybridization of the probe and the derivitized (e.g. biotinylated) target, the unhybridized target was rinsed away. The presence of the target, and therefore the tethered support, was detected by enzymatic methods involving an incubation (15 min, room temperature) with alkaline phosphatase-streptavidin (AP-SA) conjugate (5 ug/ml). The wells were washed with PBS and the presence of probe-hybridized target was detected using reagents from Enzyme Substrates, (Stafford, TX). The design of the assay system is depicted in Fig. 5. The optical density (630 nm) after 40-minutes chromogenic development at room temperature indicates the level of hybridized target as shown in Fig.6.

Claims

CLAIMS:

1. A method of attaching a molecule to a solid support, comprising: providing a molecule comprising a functional group selected from the group consisting of aldehyde, ketone and carboxyamide, providing a solid support comprising a surface having attached to the surface moieties selected from the group consisting of hydrazine, hydrazide and mixtures thereof, and contacting the surface with the molecule under alkaline conditions above about pH 7.5 to form a hydrazone bond.

2. The method of claim 1, wherein the molecule is selected from the group consisting of nucleotides, oligonucleotides, proteins, peptides, polypeptides, lipids, xenobiotics, glycoproteins, and biotin.

3. The method of claim 1, wherein said contacting takes place at a pH of about 7.5 to about 12.

4. The method of claim 1, wherein said contacting takes place at a pH of about 8 to about 10.

5. The method of any one of claims 1 to 4, wherein said contacting takes place at a temperature of about 20 to about 40 °C.

6. The method of any one of claims 1 to 5, wherein the contacting takes place in the absence of reducing conditions.

7. The method of claim 1, wherein the molecule is a 5' aldehyde-derivitized oligonucleotide.

8. The method of claim 1, wherein the molecule is a carboxyamide-derivitized biotin.

9. The method of claim 1, wherein the solid support is a hydrazine treated microtiter plate, bead, chromatography column, slide, dipstick or membrane.

10. The method of claim 1, wherein the solid support comprises a hydrazide treated surface.

11. The method of claim 1, wherein the solid support comprises a hydrazine treated surface.

12. A solid support for use in chemical or immunological assays, said support comprising a molecular probe attached via a hydrazone bond to a surface of the support.

13. The solid support of claim 12, wherein the solid support is a microtiter plate, bead, chromatography column, slide, dipstick or membrane.

14. The solid support of claim 12 comprising a glass surface.

15. The solid support of claim 12, wherein the molecular probe is selected from the group contisting of nucleotides, oligonucleotides, peptides, polypeptides, lipids, xenobiotics and biotin.

16. The solid support of claim 12, wherein the molecular probe is biotin.

17. The solid support of claim 12, wherein the molecular probe is an oligonucleotide.