Biomolecules having multiple attachment moieties for binding to a substrate surface

专利摘要:
The present invention relates to a biomolecule having a plurality of binding sites for attaching the substrate surface. Multiple attachment sites can be generated directly on the biomolecule or can be generated using branched phosphoramidite residues that can be added to form dendritic structures to provide attachment sites for substrate binding residues. . Substrate binding moieties may include non-covalent moieties. For covalent residues, oligonucleotides comprising hydrazide are provided. Such hydrazides may be introduced through a protected building block, such as phosphoramidite, or through building blocks comprising precursor forms of such hydrazides.
公开号:KR20020065644A
申请号:KR1020027008958
申请日:2000-08-11
公开日:2002-08-13
发明作者:마르쿠스 슈바이쩌；노르베르트 빈드하브；존 알. 하벤스；토마스 제이. 오노프레이；찰스 에이치. 그리프；다구앙 왕
申请人:나노겐 인코포레이티드；
IPC主号:

专利说明:

Biomolecule with multiple attachment moieties for substrate surface binding {BIOMOLECULES HAVING MULTIPLE ATTACHMENT MOIETIES FOR BINDING TO A SUBSTRATE SURFACE}
[2] The following description relates to a summary of information related to the present invention. No information provided herein is recognized as a prior art for the invention claimed herein, and any publications specifically or implicitly mentioned are not recognized as prior art for the present invention.
[3] Immobilization of oligonucleotides on substrates is an important and essential step in many fields, such as DNA chip technology, surface plasmon resonance experiments or other biosensor applications. Typically, the 3 'of the oligonucleotide with one reactive group, such as an amine, thiol or aldehyde (covalent attachment), or a stable bond forming a group, such as biotin, phenylboronic acid, etc. (non-covalent attachment) Or fix the oligonucleotide on a substrate by modifying the 5′-end. The modified oligonucleotide is then addressed to the position to be immobilized and reacted with an appropriate functional group such as aldehyde, maleimide, hydrazide, or the like, or bound with a binding molecule such as streptavidin or the like. Addressing a specific location on the substrate can be accomplished by spotting (pin or drop deposition), electronic addressing, or various other methods. In some cases, the immobilization reaction is slow and requires prolonged (overnight) incubation of the oligonucleotides on the substrate. This immobilization reaction is also reversible, allowing release of biomolecules over time.
[4] In another example, dendrimeric structures on biomolecules are described (eg, WO 99/10362, WO 96/19240, and WO 99/43287), but the use of dendrimer structures may, for example, provide a signal site for detection. It has been indicated to provide, and the biomolecule itself is simply attached to the substrate using conventional methods.
[5] In contrast, the present invention relates to an improved method of immobilizing biomolecules using oligonucleotides comprising a plurality of reactive sites, ie nucleophiles, electrophiles, and Lewis acids or bases. The advantage of this method is that the fixation rate is faster, the adhesion stability is higher, and more oligonucleotides can be immobilized on the substrate surface. This advantage is independent of the method used for immobilization. Oligonucleotides with multiple attachment sites can be obtained by both covalent and non-covalent attachment chemistry.
[6] The invention also relates to the preparation of oligonucleotides comprising one or more hydrazides. Hydrazide is a nucleophilic reactive group that can be used in any type of binding reaction. They can, for example, react with electrophilic aldehydes that form hydrazones (which may be further stabilized by reduction) and active esters that form stable covalent bonds (see FIG. 18). Such chemical methods can be used to attach fluorophores, proteins or peptides, reporter groups and other oligomers to oligonucleotides. The reaction of hydrazide can also be used to immobilize biomolecules onto a substrate. Such hydrazide modified oligonucleotides are not disclosed in the prior art.
[7] There are many advantages of the present invention disclosed herein. For example, the present invention utilizes short reaction times, allows for multiple binding sites per subject to be bound, provides stability over a relatively wide pH range, and provides adhesion in both anhydrous or aqueous conditions, An improved method of attaching molecules to any solid surface for any suitable use is provided. The present invention is useful for solid phase synthesis and / or for the synthesis of small molecule libraries such as, but not limited to, DNA, RNA, PNA, p-RNA (pyranosyl-RNA) and peptides. The present invention is also useful for analytical techniques that require immobilized reagents, including but not limited to hybridization based assays, diagnostics, and gene sequence identification.
[8] <Overview of invention>
[9] In a first embodiment of the invention, biomolecules are provided having a plurality of branched or dendrimer residues for linking functional or reactive residues that bind to the substrate surface.
[10] The use of oligonucleotides with multiple reactive sites or binders in one oligonucleotide provides significant advantages for this immobilization method. First, the use of such oligonucleotides increases the speed of the immobilization reaction. One reason for this effect is that the chance of initial contact between attachment objects by diffusion is greater when one oligonucleotide has multiple reactive sites. In addition, oligonucleotides may be immobilized via secondary and multiple covalent or non-covalent linkages formed after (or at the same time) primary linkage. However, the formation of such secondary linkages is an intramolecular reaction that is dynamically encouraged to the intermolecular primary linkage formation. This is another reason for the faster fixed speed.
[11] Second, the overall stability of the attachment increases as a number of linkages are formed between the oligonucleotide and the substrate, which is independent of the method used to contact the biomolecule with the substrate.
[12] Formation of multiple non-covalent conjugates exhibits higher overall stability of the conjugate between the oligonucleotide and the substrate and allows the use of low affinity conjugate builders due to stable immobilization. Some commonly used fixation chemistries for oligonucleotides are reversible (e.g., formation of Schiff bases between amines and aldehydes) and then require a stabilization step, for example by reduction with NaCNBH ₃ . . For such reversible reactions, immobilization via multiple linkages is advantageous because this immobilization imparts higher stability to the intermediate formed prior to the stabilization reaction. In some cases, the stability is large enough that no stabilization reaction is required.
[13] Third, the use of oligonucleotides with multiple attachment sites can produce substrates loaded with more oligonucleotides. In general, reactive sites on the substrate are generally in excess of oligonucleotides, and the adhesion enhancement due to the large number of attachment residues makes more sites available on the substrate.
[14] In other embodiments, the plurality of reactive binding moieties provided on the biomolecule can bind the biomolecule to the substrate surface in a covalent or non-covalent manner. For non-covalent bonds, a number of binding moieties may include chemical moieties such as biotin, streptavidin, phenyl boronic acid (PBA) and salicylic hydroxamic acid (SHA). For covalent bonds, a number of binding moieties may include the use of reactive hydrazide structures. Such constructs may allow for a large variety in the levels of possible binding moieties available by being branched or unbranched. Thus, in addition to providing dendritic branching structures to the biomolecules, it is also possible to branch the reactive binding moiety itself such that each branch has a reactive hydrazide member used to bind the biomolecule to the substrate surface.
[15] In other embodiments, the plurality of binding moieties on the biomolecules may cause the biomolecules attached to the substrate surface, including electronically addressable microchips, to high voltages and currents resulting from electronic biasing of the microchip electrodes. There is provided a method of protecting against accidental removal at the attachment site on the microchip. Thus, in a preferred embodiment, the present invention provides a plurality of attachment reactions that bind biomolecules to a substrate capable of withstanding a current density of at least 4 mA / cm ² .
[16] In another embodiment, the present invention provides a method for adding a reactive binding moiety to a dendritic structure attached to a biomolecule so that the addition reaction can occur in a single reaction step.
[17] In another embodiment, the present invention provides a composition of matter comprising a new chemical modification of an oligonucleotide comprising one or multiple hydrazides, thereby providing a building block (eg, a force) for the production of modified oligonucleotides. Formamidite). Such hydrazides contain reactive groups and can be used to bind oligonucleotides to fluoropores or other small molecules, peptides, proteins or antibodies, or substrate surfaces.
[18] In another embodiment, such attachment reactions can be used in methods of surface synthesis and analysis of biomolecules that require the compound to be immobilized on the surface.
[1] The present invention relates to adhesion chemistry for binding biomolecules to substrate surfaces. More specifically, the present invention relates to attachment chemistry comprising branched structures that provide biomolecules having a plurality of chemical binding moieties for binding the biomolecules to the substrate surface.
[19] 1 is a scheme illustrating a conventional method for immobilizing oligonucleotides to planar substrates. Generally, a single reactive group is used to bind the oligomer to the substrate surface.
[20] FIG. 2 is a scheme representing an immobilization method of the present invention wherein the biomolecule has a number of attachment or binding moieties that may be involved in covalently or noncovalently binding it to a substrate.
[21] 3 shows in more detail one example of a nucleic acid strand attached to a plurality of reactive residues for substrate surface binding. In this example, the chemical structure of branched phosphoramidite is added in various ways to form dendrimer structures attached to biomolecules.
[22] 4 shows a series of chemical steps to create a structure having a dendrimer structure comprising a phenyl boronic acid (PBA) attachment moiety.
[23] 5A and B show a series of chemical steps that produce chemical structures comprising oligonucleotide biomolecules having four (A) or eight (B) binding moieties that noncovalently bond biomolecules to a substrate surface.
[24] 6A-C show synthetic steps using phosphoramidite to produce biomolecules with multiple reactive sites. These moieties include ester groups, which are converted to hydrazine during deprotection of the oligonucleotides with hydrazine.
[25] FIG. 7 shows chemical structures A and B comprising biomolecules with direct attachment of hydrazide residues used to immobilize biomolecules to a substrate via covalent bonds.
[26] 8A-D show chemical synthesis that results in constructs having multiple binding moieties. In (A), branched phosphoramidite is added to the oligonucleotide, further modified with bifunctional phosphoramidite, and then deprotected with diethylamine / CH ₂ Cl ₂ and hydrazine, for substrate binding. The binding moiety having four hydrazide groups is generated. In (B), a scheme similar to (A) is provided that yields six hydrazide binding moieties. In (C), the continuous use of two different branched phosphoramidites forms 16 hydrazide binding moieties per biomolecule. In (D), branched phosphoramidite is used in two steps to form a dendrimer structure and then treated with phosphoramidite and hydrazine to form four hydrazide binding moieties per biomolecule.
[27] 9A-C show three reactions, in which Schemes A and B represent steps for preparing new phosphoramidites that are incorporated into oligonucleotide biomolecules. 9C shows a scheme in which a hydrazide-labeled oligonucleotide can be reacted with an activated-ester monomer to prepare a substrate for the immobilization of biomolecules.
[28] 10A-C are graphs of three different HPLC results of the reaction mixture for coupling hydrazide-labeled oligomers to activated ester monomers as shown in Scheme 3 in FIG. 9.
[29] 11 is a graph showing the reaction rate for the attachment of multi-labeled biomolecules. Hydrazide / N-hydroxysuccinimidyl (NHS) ester bonds are faster than the speed of other covalent bond systems and occur measurably to the speed of both non-covalent bond systems.
[30] 12 is a graph showing that the covalent attachment of labeled hydrazide oligos depends on the amount of activated ester on the substrate surface.
[31] 13 and 14 are graphs showing the ability of covalent attachment to NHS or N-hydroxy-sulfosuccinimidyl (sulfo-NHS) ester modified substrate surfaces, respectively. The graph shows specific and nonspecific fluorescence intensities from labeled oligomers attached to the electrode over the range of applied currents.
[32] FIG. 15 is a graph showing that multiple binding of hydrazide residues provides for detection of higher levels of biomolecules on a substrate.
[33] FIG. 16 is a graph showing the results of electronic reverse dot blots, where hybridization was complete only at sites containing hydrazide-modified oligonucleotides (ATA5). The capture probe specifically bound to the activated-ester-containing substrate under appropriate electronic conditions. Nonspecific captures without hydrazide did not react with the activated ester and thus could not be used for hybridization.
[34] Figure 17 shows the synthesis of hydrazide modified oligonucleotides carrying two different protocols A and B. In A, the oligomers are modified using protected hydrazide phosphoramidites and then deprotected. In B, the oligomer is modified with ester phosphoramidite and then reacted with hydrazine.
[35] 18 shows a scheme showing various functional groups that can react with hydrazide modified oligomers.
[36] 19 and 20 are examples of hydrazide oligomers that condense with aldehydes.
[37] 21 shows the recorded mean fluorescence intensity (MFI) values for oligomers labeled with 3, 4 and 8 phenylboronic acids per oligomer. The attached oligomers were subjected to intensive wash conditions to monitor the stability of the attachment system.
[38] FIG. 22 shows the dynamic equilibrium and stability of dendrimer hydrazide on an aldehyde rich permeable layer. Oligomers representing four hydrazide residues are electronically loaded onto the aldehyde rich permeable layer to form multiple hydrazone bonds. In this particular example, each bond is sensitive to hydrolysis. The stability obtained with the use of multiple attachment sites allows hydrolysis of some hydrazones, while others remain intact. Hydrazide linked through adjacent hydrazone attachment sites cannot diffuse and are thus retained in an aldehyde-rich permeable layer that can reform bonds.
[39] FIG. 23 is a graph showing the attachment of hydrazide 1, 2, 4 and 8 dendrimer oligomers on a glyoxyl agarose permeable layer coupled via hydrazone bonds.
[40] 24 is a graph showing attachment of dendrimer oligomers on acetal modified hydrogels. Acetal residues must be hydrolyzed to acids to produce aldehydes for covalent attachment capacity.
[41] FIG. 25 shows the use and improved binding of various concentrations of hydrazide oligomers on a Thermomods 3D Link® amine binding slide. FIG. 25B is an actual fluorescence image of oligomers bound to a glass slide, and their binding levels are graphically shown in FIG. 25A.
[42] FIG. 26 shows the nonspecific attachment binding level for the Thermodex slide used in FIG. 25. FIG. 26B is an actual fluorescence image of oligomers bound to a glass slide, and their binding levels are graphically shown in FIG. 26A.
[43] FIG. 27A shows a suitable pH range in which hydrazide oligomers can be successfully immobilized on a solid support, and FIG. 27B shows an improved sensitivity of hydrazide oligomers to standard amine modified oligomers detectable at lower concentrations.
[44] 28 shows one example where branched or unbranched hydrazide modified oligomers can be easily modified with other attachment systems. In this particular example, a branched oligomer having six hydrazides is modified with p-formylphenylboronic acid to prepare a branched PBA attachment probe.
[45] <Detailed Description of the Preferred Embodiments>
[46] Reference is made to certain embodiments of the invention to provide biomolecules having a plurality of substrate surface binding moieties.
[47] "Biomolecule" refers to a biologically related molecule used to contact a molecular object in a test sample. In general, biomolecules include single nucleic acids, oligonucleotides and polynucleotides, DNA, RNA, CNA (cyclohexyl nucleic acid), p-MeNA (methyl or at least partially attached to chemical moieties for binding the biomolecule to the substrate surface). Molecules such as methoxy phosphate nucleic acids), proteins, peptides, enzymes and antibodies. Biomolecules are also non-natural or synthetic molecules that are structurally derived from naturally occurring molecules such as peptide nucleic acids (PNAs) or p-RNAs (pyranosyl RNAs) attached to chemical moieties for binding the biomolecules to the substrate surface. Included. In the case of having such a binding moiety, the biomolecule may also be referred to as the "derivatized biomolecule". Thus, such biomolecules are also outlined in oxidized ribose, oligonucleotides comprising amine terminations, Hermanson, Hermanson, GT 1996, Academic Press, San Diego, CA, which is incorporated herein by reference. Any portion of a well-known biobinding pair, and / or pRNA (described in US Pat. No. 09 / 374,338, incorporated herein by reference, filed August 13, 1999, with respect to pRNA) Other nucleic acid constructs). In general, the attachment of chemical moieties to biomolecules involves covalent bonds. For attachment of derivatized biomolecules to the substrate surface, such attachment may use covalent or non-covalent bonds.
[48] "Polymer" refers to a macromolecule in which a number of small molecules, generally referred to as monomers, are assembled in series, as commonly recognized by those skilled in the art (for more detailed descriptions, see Odian, G. Principles of Polymerization, Third Edition copyright 1991 John Wiley and Sons Inc., New York, NY). In a preferred embodiment, the homopolymer may consist of a single type of monomer, while the heteropolymer consists of more than one type of monomer. In other preferred embodiments, the formation of the polymer can be achieved by thermal decomposition of the initiator (eg, AIBN, benzoyl peroxide), photolytic cleavage of the initiator (eg, UV initiation of Daracur 4265), redox reaction (eg, Polymerization only at preselected sites using cerium (IV) sulphate), ionizing radiation (eg, α, β, γ or X-rays), plasma initiation (eg argon, nitrogen, oxygen), or current This can be initiated by initiation of electrolysis using tetrabutylammonium perchlorate (Samal, SK; Nayak, BJ Polym. Sci. Polym. Chem. Ed. 1988, 21, 1035).
[49] "Binding moiety" generally refers to any chemical moiety used to attach biomolecules to a substrate surface. Binding moieties may be included in the biomolecule or may be included on the substrate surface. Table 1 binding residues provides a list of binding residues used.
[50]
[51]
[52]
[53] "Lewis base" generally means any chemical moiety capable of providing a pair of electrons to an electron deficient center. In a preferred embodiment, the Lewis base more specifically means "nucleophile", wherein the reactive center provides a pair of electrons to the carbon to form a covalent bond between the reactive center and the carbon, as recognized by those skilled in the art (enlarged) For definitions, see Smith, MB Organic Synthesis copyright 1994 McGraw Hill Inc., New York, NY, or any chemical literature).
[54] "Lewis acid" generally refers to any electron deficient chemical moiety capable of accepting a pair of electrons. "Electrophile" generally refers to the specific case where the Lewis acid is carbon, as will be appreciated by those skilled in the art (for expanded definitions, see Smith, MB Organic Synthesis copyright 1994 McGraw Hill Inc., New York, NY, Or any organic chemical literature). In a preferred embodiment, as an example, salicylic hydroxamic acid can act as a Lewis base to transfer a pair of electrons to boron, the Lewis acid of phenyl boronic acid, to form a non-covalent bond. In another preferred embodiment, as an example, hydrazide can act as a nucleophile that provides a pair of electrons to the reactive carbon center of an electrophilic NHS ester to form a covalent bond to the carbon center.
[55] "Branched linking moiety" generally refers to any chemical species that can be coupled to biomolecules through specific reactive moieties and can also be further attached to one or more molecules through other reactive centers. In a preferred embodiment, the branched linking moiety is phosphoramidite, for example as shown in Table 2, numbers 1-4. In these examples, phosphorus acts as a reactive moiety, but esters of numbers 1, 2 and 3 and protected alcohols of number 4 are other reactive centers.
[56] "Branched linking structure" generally refers to a biomolecule produced by treating a biomolecule with a branched linking moiety. Other reactive centers of branched linking moieties are contained within branched linking structures. In a preferred embodiment, as an example, branched linking structures are shown as number 5 in Table 2, wherein the biomolecules shown are the results of treatment of the biomolecules with branched linking residues, in particular the compound shown as number 4 in Table . In other preferred embodiments, the branched linking constructs can be combined with those of the same kind, wherein the biomolecules are modified with branched linking residues and then the same branched linking through the other reactive centers of the resulting branched linking constructs. Further modified by residues to form new branched linking structures. Constructing a larger branched linking structure by a series of linkages of branched linking residues can be further sustained as shown in Table 2, numbers 6-8. In another embodiment, branched linking moieties may bind to heterologous ones, wherein the biomolecules are modified with branched linking residues and then to other branched linking residues through other reactive centers of the initial branched linking residues. Further modified to form a newly branched connection structure. Constructing a larger branched linking structure by a series of linkages of branched linking residues can be further sustained, as shown in Table 2, numbers 9-12.
[57] Branched linking residues and branched linking constructs number Chemical structure Compound name or kind One Branched linking moieties: diethyl 5-{[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] methyl} isophthalate; Compound 1c; Diester phosphoramide 2 Branched linking moieties: diethyl 3-[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] glutarate; Branched diester phosphoramidite 3 Branched linking residues: dimethyl 3,3 '-(2-{[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] methyl} -2-{[2- (methoxycarbonyl) Methoxy] methyl} propane-1,3-diylbisoxy) dipropionate; Compound 1d; Tri-ester phosphoramidite
[58] 4 Branched linking moieties: 1,3-bis-((di p-methoxyphenyl) phenylmethoxy) -2-propyl O-2-cyanoethyl-N, N-diisopropylaminophosphoramidite; Symmetrically branched phosphoramidite. DMT = di- (p-methoxyphenyl) phenylmethyl 5 Branched linking structure wherein the first forming another reactive species alcohol (R = H) or protected alcohol (R = DMT) 6 Homogeneous branched linkages of the second forming another reactive species alcohol (R = H) or protected alcohol (R = DMT) 7 Third branched homogeneous branched linkage structure wherein the other reactive species are alcohols (R = H) or protected alcohols (R = DMT)
[59] 8 Homogeneous branched linkage structure of the fourth forming another reactive species alcohol (R = H) or protected alcohol (R = DMT) 9 Branched Connection Structures of Second Formation Heterogeneity 10 Branched Connection Structures of Second Formation Heterogeneity 11 Branched Connection Structures of Second Formation Heterogeneity
[60] 12 Branched Connection Structures of Third Formation Heterogeneity
[61] "Substrate" generally refers to any material having a surface comprising a moiety to which multiple reactive binding moieties of a biomolecule can be coupled. Such substrates include glass slides, functionalized glass slides, chemically activated microchip surfaces, surfaces covered with one or multiple layers of reactive molecules, or residues to which many reactive binding moieties of biomolecules can react. There may be a surface covered with a polymer. In a preferred embodiment, the substrate surface is a transmissive layer of electronically addressable microchips. In a preferred embodiment, the functional moiety, chemically active moiety or reactive moiety of the substrate is selected from, but is not limited to, the functional groups listed in Table 1.
[62] "Precursor" generally refers to any reactive moiety that can be modified with another reactive moiety by treatment with one or more chemical reagents. In a preferred embodiment, as an example, the three ester residues of 1d (number 3 in Table 2) are precursors to hydrazide. These are treated with hydrazine to transform into hydrazide residues.
[63] "Protected" generally means blocking the reactivity of the reactive moiety with one or more reagents, but the chemical reaction can be carried out at other reactive sites of the same compound without blocking or complexing from the initial reactive moiety. Upon completion of modification at another reactive site, the protecting group of the reactive moiety may be removed such that the reactive center is not blocked. In a preferred embodiment, the protected moiety is a specific type of precursor. In another preferred embodiment, as an example, the hydrazide residue of 1a of FIG. 9A is protected with a trityl group. Upon addition of 1a to the biomolecule, the trityl group is chemically removed to deprotect the hydrazide functional group.
[64] "Activatable" generally refers to any functional group that can be transformed into a reactive moiety when treated with one or more chemical reagents. "Activated" means a functional group that has undergone modification to a reactive moiety. In a preferred embodiment, the activatable moiety can be a protected moiety or precursor. In another preferred embodiment, the functional group is generally considered to be positive, nonreactive or incapable of binding to the substrate or biomolecule. When treated with one or more chemical reagents, the functional groups are modified with residues that can bind to the substrate or biomolecule. In a preferred embodiment, as an example, the ester groups of the compounds listed in Table 2, Nos. 1 to 3, are modified to hydrazide when treated with hydrazine. In another preferred embodiment, as an example, a substrate comprising an acetal group is generally considered to be nonreactive. When treated with an acid source, acetals are modified aldehydes capable of binding hydrazide modified biomolecules.
[65] By “microarray” is generally meant the geometric arrangement of a number of positions comprising the biomolecules formed, each of which is limited to a length of 1 mm or less. Microarrays include electronically addressable microarrays such as those designated "APEX chips" in US Pat. No. 5,632,957, incorporated herein by reference.
[66] Figure 2 shows the basic reaction of the overview of the invention, wherein the biomolecule is bound to the substrate surface via a number of attachment moieties. Multiple attachment moieties can be provided to the biomolecules using the following methods. Each of these methods is suitable for standard solid phase synthesis of biomolecules comprising oligonucleotides.
[67] 1. Preparation of oligonucleotides with multiple attachment sites
[68] 1.1 Synthesis of Oligonucleotides with Branched Phosphoramidites:
[69] Branched biomolecule (eg oligonucleotide) constructs with branched phosphoramidites are commercially available (Chemgenes, Ashland, MA; Glenn Research, Sterling, VA). After one or more consecutive couplings of these branched amidites in solid phase oligonucleotide synthesis (FIGS. 5A and B), oligonucleotides having two or more terminal hydroxyl groups are formed. Here, any other building block that introduces branches into oligonucleotides can be used in a similar manner. These hydroxyl groups can be reacted with a second type of phosphoramidite to form reactive groups (ie, binding moieties) for attaching biomolecules to the substrate. Such phosphoramidites are available in a number of available amidites, for example biotin amidides (eg Glenn Research, Cat No. 10595002), amino modifications (eg Glenn Research, Cat. No. 10190602), Thiol modifiers (eg, Glenn Research, Cat. No. 10192602), phenylboronic acid amidide (Prolinx, Bothell, WA) and others. It is also possible to use phosphoramidites comprising hydrazides in protected or precursor form (FIG. 9A). The result is an oligonucleotide having two or more (preferably two to eight) reactive groups.
[70] 1.2 Direct introduction of synthons with one or more reactive groups for attachment:
[71] Alternatively, biomolecules having a large number of attachment sites can be obtained by special phosphoramidite coupling. Such amidite may include one or more reactive groups for immobilization on the substrate in protected or precursor form. The reactive group in branched amidite may also be one of known functional groups such as amino groups, thiols, aldehydes or hydrazides. Examples of such amidites are shown in Figures 6A-C.
[72] 1.3 Combined Methods:
[73] A third method of synthesizing biomolecules with multiple reactive groups is a combination of branched amidite and coupling of amidite with multiple reactive sites (FIGS. 8A-C).
[74] In a particularly preferred embodiment, biomolecules are provided having bound hydrazide for attachment to the substrate surface via covalent bonds. In this embodiment, NHS and sulfo-NHS and other moieties can be used as a means to activate and couple the biomolecules of the substrate or any other type and to couple to the biomolecules or even solid surfaces. In the use of the present invention, such attachment is a novel method in which attachment of biomolecules can be performed to provide resistance to damage to bound biomolecules caused by extreme reaction conditions associated with electronic addressing of the electronic microchip. Provide a method. Accordingly, the hydrazide chemistry and many adhesion reactions of the present invention fulfill the requirements for viability in the environment of an electronic system, which requirements for water solubility of biomolecules, biomolecules and their coupling pairs on water for immobilized substrates. The need for stability, and functionality for a pH of about 4.
[75] The method used in the present invention with the addition of hydrazide binding moieties is provided in the Examples below. This example shows site specific covalent attachment of a biomolecule comprising an oligonucleotide, wherein the attachment is an N-hydroxysuccinimidyl (NHS) modified polyacrylamide permeable layer on an electronically addressable microarray. Is achieved using an electronic concentration of hydrazide modified oligomers. The hydrazide residues of the oligomers replace NHS esters that form bishydrazide bonds. Thus, this example shows the following facts. 1) Synthesis of novel hydrazide phosphoramidites (eg Compound 1) as shown in Example 1 (FIG. 9), and successful incorporation of these amidites into synthetic oligomers using standard synthetic methods, 2) preparation of an N-hydroxy- or N-hydroxysulfo-succinimidyl modified permeable layer, and 3) two layers of a permeable layer on an electronically addressable microarray (activated monomers are incorporated only in the upper layer). ).
[76] Unless otherwise indicated, all reactions are stirred with a magnetic rod. Reagents were obtained in analytical grade from Aldrich Chemical Company, Milwaukee WI, and solvents were obtained from Riedel. Column chromatography was performed using silica gel 60 (Merck, 230-400 mesh). Melting point was not corrected. IR spectra were measured on a Perkin Elmer Paragon 1000 FT-IR equipped with a Graceby Specac 10500 ATR unit. ¹ H-NMR spectra were recorded at 400 MHz using a Bruker DRX 400 spectrometer, ¹³ C spectra were recorded at 100 MHz, and ³¹ p was measured at 162 MHz. ¹ H chemical shifts were reported in units of δ using TMS as an internal standard and coupling constants were reported in units of Hz. ESI mass spectra were recorded in negative ionization mode on a Finnigan LCQ instrument.
[77] <Example 1>
[78] Experimental Example 1.1
[79] Synthesis of N-triphenylmethyl-6-hydroxycaproic acid hydrazide (Compound 5, FIG. 9A) :
[80] To a solution of 6.2 g (20 mmol) of tritylhydrazine hydrochloride (3a) in 200 ml of THF was added 2.22 g (22 mmol, 1.1 equiv) of triethylamine. The solution was stirred at rt for 15 min, filtered and concentrated to afford compound 3, then treated with 2.29 g (20 mmol, 1 equiv) of ε-caprolactone (compound 4 ). The mixture was heated to 65 ° C. for 5 hours and then cooled to room temperature for 18 hours. The precipitate was collected and recrystallized from ethyl acetate to give 3.55 g (45%) of a white powder (Compound 5).
[81]
[82] Experimental Example 1.2
[83] Synthesis of 6-[(2-cyanoethoxy (diisopropylamino) phosphanyloxy] -N'-tritylhexanohydrazide (Compound 1a , FIG. 9A) :
[84] 4.0 g (31 g) of N-ethyldiisopropyl amine over 15 minutes in a solution of 3.0 g (7.7 mmol) of N-triphenylmethyl-6-hydroxycapron hydrazide (Compound 5) in 50 ml of anhydrous dichloromethane at room temperature mmol, 4 equiv) and 2.01 g (8.5 mmol, 1.1 equiv) of chloro (diisopropylamino) -β-cyanoethoxyphosphine (Compound 6) were added slowly. After complete addition, the reaction was stirred for 1 hour, concentrated and chromatographed (ethyl acetate / n-heptane 2/3 with 0.2% triethylamine) to give 3.19 g (70%) of 1a as a pale yellow foam.
[85]
[86] Experimental Example 1.3
[87] Preparation of ethyl 6-[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] hexanoate (Compound 1b , Fig. 9B, Scheme 2) :
[88] 5.17 g (40 mmol, 4 equivalents) of N-ethyldiisopropyl amine over 15 minutes in a solution of 1.65 g (10 mmol) (Compound 7) of ethyl 6-hydroxyhexanoate in 30 ml of dichloromethane at room temperature over 15 minutes 6 2.6 g (11 mmol, 1.1 equiv) were added slowly. After complete addition, the reaction was concentrated by further stirring for 15 minutes and chromatographed (ethyl acetate / n-heptane 1/4 with 0.2% triethylamine) to give 2.47 g (69%) of compound 1b as a clear oil. .
[89]
[90] Experimental Example 1.4
[91] Ester phosphoramidite: Preparation of diethyl 5-{[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] methyl} isophthalate (Compound 1c, FIG. 6B) :
[92] N- over 15 minutes in a solution of 1.29 g (5 mmol) (98%, Aldrich; CAS 181425-91-2) in diethyl 5- (hydroxymethyl) isophthalate [252.27] in 20 ml of anhydrous dichloromethane at room temperature. 2.59 g (40 mmol, 4 equiv) of ethyldiisopropyl amine [129.25] and 2-cyanoethyl N, N-diisopropyl-chloro-phosphoramidite [236.68] (Aldrich; CAS 8999270-1) 1.3 g (11 mmol, 1.1 equiv) was added with stirring. The mixture was concentrated and the salt precipitated with 30 mL of ethyl acetate / n-heptane (2: 3). The hydrochloride precipitate was filtered off, the filtrate was concentrated and added directly to the chromatography column. Elution with ethyl acetate / n-heptane (1: 4) containing several drops of triethylamine gave 1.6 g (70%) of 1c as a colorless oil.
[93]
[94] Experimental Example 1.5
[95] Dimethyl 3,3 '-(2-{[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] methyl} -2-{[2- (methoxycarbonyl) ethoxy] methyl} propane Synthesis of -1,3-diylbisoxy) dipropionate (Compound 1d , FIG. 8B) :
[96] Tris-2,2,2-{[(methoxycarbonyl) ethoxy] methyl} ethanol (CAS 169744-28-9; (Coutts, S .; Jones, DS; Livingston, in 2 ml of anhydrous dichloromethane at room temperature) DA; Yu, L .: 1995, Chemically-defined nonpolymeric valency platform molecules and conjugates, European patent application EP 0642798A2) 0.4 M solution of 1 H-tetrazole in anhydrous acetonitrile (solid DNA synthesis) in 300 mg (0.760 mmol) of solution 2 drops of standard activator solution from) and 274 mg (0.91 mmol; 1.1 equiv. 2-cyanoethyl N, N, N ', N'-tetraisopropylphosphorodiamidite (Aldrich; CAS 102691-36-1) ), And by TLC, stirred at room temperature until the starting material was completely consumed (3 hours) The solvent was removed in vacuo and the residue was purified by silica gel chromatography containing several drops of triethylamine Elution with ethyl acetate / n-heptane (2: 3) afforded 240 mg (53%) of a colorless oil 1d .
[97]
[98] Experimental Example 1.6
[99] Synthesis of oligonucleotides with trityl protected hydrazide amidite (e.g. Compound 1 ; see FIG. 17A) :
[100] Oligonucleotides were synthesized using solid phosphoramidite chemistry on an automated oligonucleotide synthesizer. Phosphoramidite with protected hydrazide was added to a 0.1 M solution in acetonitrile and continuously coupled to the desired position using standard activation reagents and coupling times.
[101] CPG bound oligos (1 mmol) were placed in a 1.5 ml test tube and treated with 2.0 ml of concentrated NH ₄ 0H. After 2 hours at 55 ° C., the ammonia solution was removed and evaporated to dryness under reduced pressure. The residue was dissolved in 1 ml of water and filtered through a 0.45 μm syringe filter. By reversed phase HPLC using Merck LiChrospher RP 18, 10 μΜ, column (analysis: 4 x 250 mm, flow rate = 1.0 ml / min; preparative: 10 x 250, flow rate = 3.0 mL / min) Trityl protected hydrazide oligos were purified using 0.1 M triethylammonium acetate pH = 7.0 (TEAA) with buffer A and 75% acetonitrile in buffer A with buffer B. A gradient of 0% B to 100% B within 100 minutes was used for analysis and preliminary separation. Fractions containing trityl on the product were pooled and evaporated to dryness.
[102] To remove the trityl protecting group, the oligos were treated with 80% acetic acid for 30 minutes at room temperature. The acid was removed in vacuo, the residue dissolved in water and then extracted twice with ethyl acetate. The aqueous layer was dried again and redissolved. HPLC analysis generally shows a single product (in some cases as a double peak), which can be used in further reactions without purification. Alternatively, HPLC purification can be performed using the solvent system described above.
[103] Experimental Example 1.7
[104] In situ production of hydrazide functional synthesis of oligonucleotides using phosphoramidite containing precursor forms (e.g. , esters such as compound 1b , FIG. 9B, Scheme 2; see FIG. 17B) :
[105] Oligonucleotides were synthesized using solid phosphoramidite chemistry in an automated oligonucleotide synthesizer. Phosphoramidite, having the precursor form of hydrazide, was added to a 0.1 M solution in acetonitrile and continuously coupled at the desired position using standard activation reagent and coupling time. The precursor form of hydrazide is stable to the conditions of oligonucleotide synthesis, but since reactive hydrazide is not formed until incubation with hydrazine, phosphoramidite containing hydroxyl groups labeled with an acid labile protecting group And hydrazide precursors can be used to introduce hydrazide anywhere in the oligonucleotide.
[106] CPG bound oligos (1 mmol) were treated with a solution of 50 mg of diethylamine in 3.5 mL of dichloromethane. After overnight incubation (light block), the supernatant was removed and the oligos bound to the support were washed several times with dichloromethane and dried in vacuo.
[107] For cleavage of the benzoyl and isobutyryl protecting groups, conversion of the oligo's 5'-terminated ester to hydrazide, and cleavage of the oligo from the support (FIG. 17B), 1 ml of 24% hydrazine hydrate of CPG with bound oligos Treated with. Stirring was continued at 4 ° C. for 18 hours and then the reaction was completed. Separation of oligos from hydrazine solution could be achieved by reverse phase extraction (eg Sep-Pak or HPLC).
[108] The C18 Sep-Pak cartridge (0.5 g Waters, No. 20515) was activated by washing with 10 ml of acetonitrile followed by 0.1 M triethylammonium bicarbonate buffer pH 7.0 (TEAB). The hydrazine solution was diluted with 5-fold volume of TEAB and added to the cartridge. The oligos were bound to a Sep-Pak column and the residual hydrazine was removed by washing with 10 mL of TEAB. The oligo was then eluted from the column using TEAB / acetonitrile (1: 2). The oligo containing fractions were pooled and evaporated to dryness. For RP-HPLC characterization and purification of the product, the same conditions as described in protocol 1 could be applied.
[109] Other examples are provided below, wherein the oligomers are processed and linked to multiple attachment moieties of the invention. Oligos were numbered in their respective order of description in this disclosure.
[110] Example 2:
[111] Experimental Example 2.1 Synthesis of Unbranched Oligonucleotides
[112] 2.1.1 Oligos 9 Hydrazide-15mer: (1-TTT TTT TTT TTT TTT-3 ')
[113] Synthesis and deprotection was performed as described for amidite compound 1a . Trityl ON product eluted at 42.2 minutes under the conditions described. Oligo 9 eluted at 25.6 min (double peak). LRMS (ESI): M Theoretical: 4709.15. Found: 4709.5.
[114] 2.1.2 Oligos 10 Hydrazide-19mer: (1-dGA TGA GCA GTT CTA CGT GG-3 ')
[115] Synthesis and deprotection was performed as described for amidite compound 1a . Trityl ON product eluted at 41.5 minutes under the conditions described. Oligo 10 eluted at 25.1 minutes (single peak). HRMS (ESI): M Theoretical: 6092, Found: 6092.
[116] 2.1.3 In-situ creation of hydrazide 11 ; Hydrazide 19mer (8-dGA TGA GCA GTT CTA CGT GG-Cy3)
[117] Synthesis of oligonucleotides was performed as described above. A fluorophor was labeled at the 3 'end of the oligo using a CPG support loaded with Cy3 dye. CPG bound oligos were treated as described in Example 1 (E) above and the product was purified by RP-HPLC. The hydrazide oligo eluted at 31.8 min under the HPLC conditions described in Example 1 (D). LRMS (ESI): M Theoretical: 6599.7, Found: 6598 ± 2.
[118] Experimental Example 2.2 Synthesis of Branched Oligonucleotides
[119] To introduce multiple hydrazides into oligonucleotides, branched phosphoramidites, phosphoramidites having at least one ester group converted to hydrazide, and combinations of the two were used. In this way oligonucleotides containing one to several (up to 40) hydrazides could be synthesized. Such experiments have been described using p-RNA and are applicable to other oligonucleotides such as DNA.
[120] Experiment 2.2.1 Synthesis of p-RNA Oligonucleotide
[121] With the exception of the following exceptions and modifications, the synthesis of p-RNA oligonucleotides is described by Miculka, C .; Windhab, N .; Brandstetter, T. Burdinski, G; PCT patent application No. WO 99/15540 (1999). )). The phosphoramidite of pentopyranosyl nucleoside was dried in vacuo using KOH and dissolved in anhydrous acetonitrile to give a 0.1 M solution. This solution was dried for 3 hours on freshly activated molecular sieves (3x3) and then used for solid oligonucleotide synthesis on a PE Biosystems Expedite 8905 DNA synthesizer. The other phosphoramidite was dissolved in 0.1 M in anhydrous acetonitrile and used without further treatment. For p-RNA oligonucleotides carrying a Cy3 dye at the 2'-terminus, the CPG support custom was loaded with monomethoxytrityl protected Cy3 (CAS: 182873-80-9, AP-Biotech, Freiburg, Germany) 0.1 M solution of anhydrous pyridinium hydrochloride in anhydrous acetonitrile was used as activator. Detritylation time for pentopyranosyl nucleosides was increased to 10 minutes and coupling time was increased to 25 minutes. All other reagents and solutions and methods are according to the instrument manufacturer's recommendations.
[122] Experiment 2.2.2 Deprotection of p-RNA Oligonucleotides:
[123] For β-cyanoethyl protecting group cleavage, the oligonucleotides were treated with a 1.5% (w / v) solution of diethylamine in dichloromethane overnight at room temperature (light blocking). The supernatant was removed and the support bound oligonucleotides were washed several times with dichloromethane and dried in vacuo.
[124] For cleavage of benzoyl and isobutyryl protecting groups, conversion of the 5 'end of the oligo ester to hydrazide, and cleavage of the oligo from the support, CPG with bound oligos was treated with 1 ml of 24% hydrazine hydrate. After 18 hours under constant stirring at 4 ° C., the reaction was complete. Separation of oligos from hydrazine solution can be accomplished by reverse phase extraction (eg Sep-Pak or HPLC).
[125] The C18 Sep-Pak cartridge (0.5 g Waters, No. 20515) was activated by washing with 10 mL of acetonitrile followed by 10 mL of 0.1 M triethylammonium bicarbonate buffer pH 7.0 (TEAB). The hydrazine solution was diluted with 5 volumes of TEAB and added to the cartridge. After the oligos were bound to the Sep-Pak column, residual hydrazine was washed away using 10 mL of TEAB. The oligo was then eluted from the column using TEAB / acetonitrile (1: 2). The oligo containing fractions were pooled and evaporated to dryness. Characterization and purification of the product were carried out using Merck LiChrospher RP 18, 10 μΜ, column (analytical: 4 x 250 mm, flow rate = 1.0 ml / min; preparative: 10 x 250, flow rate = 3.0 mL / min). Reverse phase HPLC was used to achieve 0.1 M triethylammonium acetate pH = 7.0 (TEAA) as buffer A and 75% acetonitrile in buffer A as buffer B. A gradient of 0% B to 100% B in 100 minutes (HPLC Method A) or 30 minutes (HPLC Method B) was used for analysis and preliminary separation.
[126] A. Oligo 12 : Cy3 labeled p-RNA oligo with one hydrazide: p-RNA oligo 4 '-(Hyd _One) TAG GCA TT (Cy3) -2 '
[127] Synthesis and deprotection was performed as described for amidite compound 1b .
[128] B. Oligo 13 : Cy3 labeled p-RNA oligo with three hydrazides: p-RNA oligo 4 '-(Hyd ₃) TAG GCA TT (Cy3) -2 '
[129] Synthesis and deprotection was performed as described for amidite compound 1d . Under the conditions described, the product eluted at 37.9 minutes (HPLC method A). LRMS (ESI): M Theory: 3516.6. Found: 3515.
[130] C. Oligos 14 : Cy3 labeled p-RNA oligo with 4 hydrazides: p-RNA oligo 4 '-(Hyd ₂) ₂(SBA) TAG GCA TT (Cy3) -2 '
[131] Synthesis and deprotection was performed as described for amidite compound 1c and symmetrically branched phosphoramidite (SBA; Clontech, No. 5252-2). Under the conditions described, the product eluted at 37.3 minutes (HPLC method A). LRMS (MALDI): M Theory: 3784.7. Found: 3784.
[132] D. Oligo 15 : Cy3 labeled p-RNA oligo with 8 hydrazides: p-RNA oligo 4 '-(Hyd ₂) ₄(SBA) ₂(SBA) TAG GCA TT (Cy3) -2 '
[133] Synthesis and deprotection was performed as described for amidite compound 1e and symmetrically branched phosphoramidite (SBA; Clontech, No. 5252-2). Under the conditions described, the product eluted at 36.9 minutes (HPLC method A). LRMS (MALDI): M Theory: 4661.1. Found: 4464.
[134] E. Oligos 16 : Cy3 labeled p-RNA oligo with spacer and 8 hydrazides: p-RNA oligo 4 '-(Hyd ₂) ₄(SBA) ₂(SBA) (S18) TAG GCA TT (Cy3) -2 '
[135] Synthesis and deprotection was performed as described for amidite compound 1c and symmetrically branched phosphoramidite (SBA; Clontech, No. 5252-2) and spacer 18 (S18, Glen research No. 101918-02). . Under the conditions described, the product eluted at 38.7 minutes (HPLC method A).
[136] F. Oligo 17 : Cy3 labeled p-RNA oligo with 16 hydrazides: p-RNA oligo 4 '-(Hyd ₂) ₈(SBA) ₄(SBA) ₂(SBA) TAG GCA TT (Cy3) -2 '
[137] Synthesis and deprotection was performed as described for amidite compound 1c and symmetrically branched phosphoramidite (SBA; Clontech, No. 5252-2). Under the conditions described, the product eluted at 38.7 minutes (HPLC method A).
[138] G. Oligo 18 : P-RNA oligo with 4 hydrazides (without Cy3 dye): p-RNA oligo 4 '-(Hyd ₂) ₂(SBA) TAG GCA TT-2 '
[139] Synthesis and deprotection was performed as described for amidite compound 1c . Under the conditions described, the product eluted at 12.75 minutes (HPLC method B). LRMS (ESI): M Theoretical: 3275.1. Found: 3275.4.
[140] Experimental Example 2.3
[141] General method of converting hydrazide oligonucleotides to boronate oligonucleotides
[142] 50 nmol of hydrazide oligonucleotide is dissolved in 200 μl of 10 mM ammonium acetate buffer, pH 4.0, 4-formylphenylboronic acid per hydrazide (Aldrich No. C43,196-6; CAS: 87199-17-5) 15 equivalents was added. For oligonucleotides containing four hydrazides, 30 μl of a 0.1 M solution of 4-formylphenylboronic acid in DMSO (3 μmol) was used, for example. The mixture was incubated for 1 hour at room temperature, 20 equivalents of NaCNBH ₃ per 4-formylphenylboronic acid was added, and the incubation was continued for 1 hour at room temperature. For example, for oligonucleotides with four hydrazides, 150 μl (150 μmol) of 1 M NaCNBH ₃ solution (6.3 mg dissolved in 1 ml) in 10 mM ammonium acetate buffer (pH 4.0) is required.
[143] Removal of excess 4-formylphenylboronic acid and sodium cyanoborohydride is removed by the method of HPLC, gel filtration (Pharmacia PD10 column), or solid phase extraction (Merck LiChrolute column). For boronate modified oligonucleotides it is important to use end capped HPLC columns. Typical conditions are Phenomenex Luna phenyl hexyl column (analysis: 4.6 x 250 mm, flow rate = 1.0 ml / min; preparative: 10 x 250, flow rate = 3.0 mL / min), 0.1 M in buffer A 75% acetonitrile in Buffer A is used with triethylammonium acetate pH = 7.0 (TEAA) and Buffer B. A gradient of 0% B to 100% B in 100 minutes (HPLC Method A) or 30 minutes (HPLC Method B) was used for analysis and preliminary separation. Fractions containing product were pooled and evaporated to dryness.
[144] A. Oligo 19 : P-RNA oligo with one boronate: p-RNA oligo 4 '-(PBA) TAG GCA TT (Cy3) -2'
[145] Synthesis and deprotection was performed as described in the general protocol using oligonucleotide 12 as starting material.
[146] B. Oligo 20 P-RNA oligos with three boronates: p-RNA oligo 4 '-(PBA) ₃TAG GCA TT (Cy3) -2 '
[147] Synthesis and deprotection was performed as described in the general protocol using oligonucleotide 13 as starting material.
[148] C. Oligos 21 P-RNA oligos with four boronates: p-RNA oligo 4 '-(PBA) ₄(SBA) TAG GCA TT (Cy3) -2 '
[149] Synthesis and deprotection was performed as described in the general protocol using oligonucleotide 14 as starting material.
[150] D. Oligo 22 : P-RNA oligo with 8 boronates: p-RNA oligo 4 '-(PBA) ₈(SBA) ₂(SBA) TAG GCA TT (Cy3) -2 '
[151] Synthesis and deprotection was performed as described in the general protocol using oligonucleotide 15 as starting material. Under the conditions described, the product eluted at 46.3 minutes (HPLC method A).
[152] E. Oligos 23 : P-RNA oligo with spacer 18 and 8 boronates: p-RNA oligo 4 '-(PBA) ₈(SBA) ₂(SBA) TAG GCA TT (Cy3) -2 '
[153] Oligonucleotide 16 was used as starting material to perform synthesis and deprotection as described in the general protocol.
[154] F. Oligo 24 : P-RNA oligo with 16 boronates: p-RNA oligo 4 '-(PBA) ₁₆(SBA) ₄(SBA) ₂(SBA) TAG GCA TT (Cy3) -2 '
[155] Synthesis and deprotection was performed as described in the general protocol using oligonucleotide 17 as starting material. Under the conditions described, the product eluted at 49.0 minutes (HPLC method A).
[156] G. Oligo 25 : P-RNA oligo with one boronate: p-RNA oligo 4 '-(PBA) TAG GCA TT (Cy3) -2'
[157] Synthesis and deprotection was performed as described in the general protocol using oligonucleotide 18 as starting material.
[158] Example 3: HPLC Analysis
[159] After completion of the synthesis of hydrazide oligos, the solution reaction kinetics of hydrazide labeled oligos with NHS or sulfo-NHS esters were examined by the first set of experiments. 5 μl of 10 mM NHS acrylate was added to a solution of 5 μl of 132 μM hydrazide ATA5 in 30 μl of 50 mM histidine. The solution was stirred for a short time at room temperature and then injected into an HPLC system. HPLC results of the compound in solution showed the amount of hydrazide ATA5 and N'-acrylo-ATA5 dihydrazide present in the reaction mixture for a given reaction time. The retention times of starting ATA5 hydrazide and modified ATA5 hydrazide were different and could be distinguished.
[160] 10A-C show three separation records of the reaction mixture. The first recording (A) was obtained from unmodified ATA5 hydrazide (A) and the third recording (C) was used to prepare a fully modified ATA5 hydrazide (B) after 5 minutes of reaction time using NHS acrylate. Indicates. Interim record (B) shows incomplete formula captured in reaction for 1 minute. Assuming approximate consumption of ATA5 hydrazide, a pseudo-first order reaction rate of 1200 M ⁻¹ s ⁻¹ is determined.
[161] A comparison of this speed with other attachment systems used is shown in FIG. 11. The reaction rate for NHS esters with hydrazide in aqueous environment represents a very effective reaction. In addition, the pH of the reaction was varied to determine the pH dependence of the hydrazide formula. Experiments were performed using a 50 mM buffer system adjusted to pH 6, 5.5, 5.0, 4.5 and 4 using HCl. However, at pH 4, hydrazide oligos were unaffected and showed no modifications, thus exhibiting a pH lower limit of about 4.5.
[162] Example 4: Chip Fabrication
[163] Chips containing microarrays were treated with plasma for 5 minutes under argon. Subsequently, 25 sites of 1 cm × 1 cm chips were silyzed using vapor phase deposition. To the center of the microarray was added 0.10 μl of a 20 (weight)% solution of 9: 1 (molar ratio) acrylamide / bisacrylamide in 1: 1 DMSO / H ₂ O with 0.3% Daracur 4265 as the UV initiator. The chip was placed in a microreaction template system and the microarray site was pressed against a UV window containing a 4 μm ² cavity (3 mm on the side). The solution was irradiated with UV light for 20 seconds, removed from the mold system, washed with water and dried with air. The wells formed a square hydrogel layer on the microarray. Excessive polymerization beyond the parameters of the template was removed.
[164] To the permeable layer present 0.80 μl of a solution containing 20 (weight)% monomer concentrations of NHS or sulfo NHS / Am / Bis 10/83/7 (molar ratio) was added and the polymer present was saturated for 1 minute. The chip was loaded onto a microreaction mold system and polymerized as above using a circular mold having a diameter of 4.6 mm and a well depth of 5 μm. This second mold completely contained the existing rectangular layer and extended beyond it. Attachment of the second layer was achieved through the insertion of polymer chains and bonding silanes. The chips were washed with water, dried using compressed air and then tested in the following experiments.
[165] Experimental Example 4.1: Activated ester concentration; Marked capture address
[166] 500 hydrazide-T12-BTR was electronically loaded with a specific labeled capture on a chip with two layers of permeable layers comprising 0, 1, 2 and 4% sulfo-NHS as described above and 50 mM nM biotin T12-BTR was used as a nonspecific labeled capture. All solutions were buffered in 50 mM histidine. The capture was addressed to 4 pads at a time for 120 seconds with a current of 500 nA / pad. Each chip was washed with 1% SDS, 0.2 x STE and soaked in 1% SDS for 20 minutes. The chip was imaged for 1 second and the average MFI value was recorded.
[167] As can be seen in FIG. 12, the covalent attachment of the labeled hydrazide oligo depends on the amount of activated ester in the permeable layer and increases with increasing concentration. Nonspecific attachment of biotin labeled oligos is also quite small, with an average of 40 MFI / s in the experiment.
[168] Experimental Example 4.2: Electronic Conditions
[169] As described above, 500 and 5 nM hydrazide-T12-BTR were electronically loaded as specific labeled captures on a chip to which a two-ply transmissive layer comprising 10% NHS or sulfo-NHS was connected. 500 mM nM Biotin-T12-BTR was used as a nonspecific labeled capture. All solutions were buffered in 50 mM histidine. Captures were addressed 3 pads at a time for 120 seconds with currents of 400, 500, 600, 700 and 800 nA / pad. Nonspecific capture was loaded at 800 nA / pad. Each chip was washed with 1% SDS, 0.2 x STE and soaked in 1% SDS for 20 minutes. The chip was imaged for 1 second and the average MFI value was recorded.
[170] As can be seen in FIG. 13, the attachment of specific traps to the NHS modified permeable layer increased significantly at 600 nA, but the sulfo-NHS modified hydrogel required slightly higher current for maximum attachment. (FIG. 14).
[171] Experimental Example 4.3: Effect of Multiple Binding
[172] A 3-ply permeable layer comprising 10% NHS was loaded with a Cy3 labeled ATA5 oligo comprising 1, 2, 4 or 8 hydrazide residues as described above. Four oligomers were electronically addressed for 120 seconds at a current of 700 or 800 nA / pad at 500 nM and buffered in 50 mM histidine. After completion, the chips were washed and binding levels were measured.
[173] The recorded MFI / s values are shown in FIG. 15. Comparison of the number of hydrazide residues available for attachment per oligomer at the same current shows increased levels of binding with increasing hydrazide to the oligomer.
[174] Experimental Example 4.4 Reverse Dot-Blot Electronic Hybridization
[175] Two-layered permeable layers comprising 15% NHS were loaded with octa-hydrazide ATA5 oligomers having Cy3 labels as specific captures as described above. Specific captures were loaded for 120 seconds at a current of 600 or 700 nA / pad at 500 nM and buffered in 50 mM histidine. Electronic hybridization was performed using 5 nM RCA5 T12-Cy5 as a specific target and a solution of 5 nM RCA4-Cy5 was used as a nonspecific target. Targets were loaded for 60 seconds at 400 nA / pad and chips were washed and imaged according to standard protocols.
[176] The data provided in FIG. 16 mainly shows hybridization of specific targets to nonspecific targets. It should also be noted that this is consistent with the reported data, with an increase in current of 600 to 700 nA for the electronic loading of the capture increases hybridization.
[177] Example 5: Synthesis of Biomolecules Having Non-Covalent Residues
[178] 4 and 5A and B show the synthesis of oligonucleotides comprising a plurality of binding moieties. In Fig. 4, oligo synthesis is shown where a single branched phosphoramidite comprising two PBAs is added. 5A and B show two branches with four PBAs and three branches with eight PBAs, respectively. Synthesis shown was performed on an ABI394 DNA synthesizer. The stepwise coupling yield of branched phosphoramidite was about 96-98% similar to normal nucleotide phosphoramidite. PBA phosphoramidite was added in the last step. Cleavage of the oligonucleotides from the solid support and removal of the protecting groups was the same as handling ordinary oligonucleotides as is well known to those skilled in the art.
[179] PBA-containing branched oligonucleotides were purified and analyzed by HPLC. HPLC of PBA-containing oligonucleotides showed broader peaks than those of normal oligonucleotides.
[180] Experimental Example 5.1: Electronic Loading of Biomolecules Through Non-Covalent Residues
[181] 20 nM unbranched and branched PBA-containing ATA5 capture probes were electronically loaded onto the hydrogel substrate. Capture probes were loaded with 10 pads at a time in 50 mM histidine for 120 seconds. 20 nM RCA5-BTR was manually loaded for 5 minutes. The substrate was washed and imaged. Analysis revealed that both branched and unbranched capture probes were anchored to the permeable layer as desired.
[182] Experimental Example 5.2 Stability of Electronically Loaded Biomolecules Through Non-Covalent Binding Residues
[183] Oligos 20, 21 and 22 (p-RNAs containing 3, 4 and 8 PBA binding sites) were electronically addressed to SHA modified hydrogel chips. At the end, initial images were recorded after the standard washing method described above. The chip array was then washed regularly by repeated washing with 10 μl of 50 mM histidine. Images were recorded after five washes. The results shown in FIG. 21 include two features. First, the recorded signal for higher grade dendrimers with a higher number of attachment sites per oligosaccharide is significantly higher. In addition, the signal is quite stable over 25 wash cycles, indicating an improved stability due to the use of the dendrimer attachment system. Oligo 22 lost about 14% of its initial signal and oligos 20 and 21 decreased by 25 and 35%, respectively.
[184] Example 6: Covalent Attachment Through Multiple Hydrazone Formations
[185] In advance, oligos modified with a single amine or hydrazide were electronically loaded onto an aldehyde modified hydrogel. The interaction of aldehydes with amines or hydrazides forms imines (carbons with double bonds to nitrogen) or hydrazones, respectively. These residues are reversible under aqueous conditions and must be further reduced with NaBH ₃ CN to form stable irreversible covalent bonds. In addition, electronic concentration oligomers comprising a single hydrazide attached the oligomer to the substrate through hydrazone formation. Removal of the reduction step resulted in the formation of unstable bonds that readily hydrolyzed, wherein the bound oligos were readily diffused and removed. If there is a significant number of hydrazones formed per oligo, the use of dendrimer hydrazide provides a method of covalent attachment via somewhat labile bonds that do not require further reduction. Reversible hydrazone formation may occur at some binding sites, while other sites remain intact (FIG. 22). Hydrazide cannot diffuse, is trapped in an aldehyde-rich environment, and can be easily reformed. This equilibrium uses an increased number of attachment sites per oligo and is believed to provide a stable attachment system unless all bonds are hydrolyzed immediately. The aldehyde-rich permeable layer may be prepared directly as in glyoxyl agarose or may be formed from an acetal modified permeable layer. In the latter, the acetal moiety readily hydrolyzes in the presence of an acid to form an aldehyde. Acetals act as protecting groups, preserving aldehyde functionality until activation is required. Hydrolysis can be completed by exposure to acid solution for 1 hour or by treatment with a weak electronic flow buffered in dilute salt solution. The latter method provides site specific hydrolysis by using an acid generated at the cathode.
[186] Experimental Example 6.1: Dendrimer hydrazide oligomer attached to glyoxyl agarose
[187] The standard 25 site chip was spin coated with glyoxyl agarose (FMC, Princeton, NJ). 500 nM hydrazide Cy3 labeled oligos comprising 1, 2, 4 and 8 hydrazides were loaded electronically at 500 nA / pad for 2 minutes each and buffered in 50 mM histidine. Chips were washed and imaged according to established methods. The recorded MFI / s values are shown in FIG. Oligos with one or two hydrazides were quite unstable and showed little or no detectable fluorescence above background noise as expected. Oligos with a higher number of hydrazides could form stable covalent bonds.
[188] Experimental Example 6.2: Dendrimer hydrazide oligomers attached to acetal modified hydrogels; Deprotection and Covalent Bonding
[189] Standard 25 array site microchips were modified with single layer hydrogels consisting of acrylamide, bisacrylamide and vinylacetal in a 15: 2: 3 ratio. Selected sites were treated with a current of 300 nA / pad in 50 mM NaCl solution for 2 minutes to hydrolyze the acetal functional groups that expose aldehydes. Dendrimer hydrazide oligomers containing 8 hydrazides per oligo were loaded electronically at 500 nA / pad for 2 minutes and buffered in 50 mM histidine for activated and unactivated pads. Nonspecific oligos were also loaded electronically at both acetal and aldehyde modified sites. After a standard wash cycle, the chips were imaged. Recorded MFI / s data is shown in FIG. 24.
[190] As can be seen in FIG. 24, the pad electronically loaded with the dendrimer labeled oligomer after being electronically activated showed the highest fluorescence signal. Interestingly, the pads remaining as acetals, which were not pre-addressed, also showed some attachment of hydrazide modified oligomers. Perhaps the electronic flow applied to concentrate the oligomers locally generated enough acid to exceed the buffering capacity of histidine and thus could hydrolyze significant amounts of acetal residues.
[191] Example 7: Coupling of hydrazide oligonucleotides to molecules other than the substrate surface
[192] Experimental Example 7.1 Reaction of hydrazide-15mer 9 with benzyloxy acetaldehyde: FIG. 19
[193] 10 μmol of hydrazide oligonucleotide 9 was dissolved in 60 μl of 10 mM ammonium acetate buffer (pH 4.0). One drop of benzyloxyacetaldehyde (CAS: 6065-87-3; C ₉ H ₁₀ O ₂ [150. 1760] Aldrich No. 38, 218-3) was added and the mixture was allowed to stand at room temperature for 1 hour. Solvent and excess aldehyde was removed in vacuo and the product was purified by HPLC (column: Merck LiChrospher RP 18, 10 μΜ, 4 x 250 mm; buffer A = 0.1 M triethylammonium acetate pH = 7.0, buffer B = buffer A 75% acetonitrile in; flow rate = 1.0 mL / min; gradient: 0% B to 100% B) in 100 minutes. The retention time of the product was 30.7 minutes and the oligo 9 eluted at 25.5 minutes.
[194] Experimental Example 7.2 Binding Reaction of Oligo 10 and Peptides, Fig. 20
[195] Oligo 10 4.4 nmol was dissolved in 60 μl 10 mM ammonium acetate buffer (pH 4.0). 44 nmol (10 equiv) of antipain hydrochloride (CAS: 37682-72-7; C ₂₇ H ₄₄ N ₁₀ 0 ₆ ㆍ 2HCl; [677.6304]; Calbio No. F178220) in 15 μL of buffer was added and at room temperature Stir for 3 hours. The intermediate product was reduced to NaBH ₃ CN (100 equiv) for 1 hour at room temperature. The product was purified by HPLC (column: Merck LiChrospher RP 18, 10 μΜ, 4 x 250 mm; buffer A = 0.1 M triethylammonium acetate pH = 7.0, buffer B = 75% acetonitrile in buffer A; flow rate = 1.0 mL / min; Gradient: separated by 10% B to 85% B) within 60 minutes. Retention time of the product (oligonucleotide peptide conjugate) was 16.5 minutes and oligo 10 eluted at 13.9 minutes. MS (ESI): theory: 6680.6; Found: 6679.6.
[196] Example 8: Manual Application of Hydrazide Modified Biomolecules on Slide Surface
[197] When binding hydrazide modified oligonucleotides to commercial slides, a series of p-RNA oligonucleotides containing 1 to 16 hydrazides was used. With oligonucleotides 12, 13 and 14, oligomers having three and six hydrazides prepared from 1d were used. In addition, amine terminated oligomers (prepared with 5 ′ amino modifications C6; Glenn Research) and unmodified oligonucleotides were used as nonspecific controls. All oligomers were labeled with Cy3 at the 2 'end and had the same nucleotide sequence.
[198] Experimental Example 8.1: Attachment to Surmodics 3D Link® Amine Bonding Slide
[199] Oligonucleotides were dissolved at pH 8.5 in concentrations ranging from 10 μM to 100 nM in 3D Link® print buffer (Surmodics, Inc, Eden Prairie, Minnesota). From each solution, 0.5 μl was added directly to the slide surface and incubated at room temperature in a chamber sealed with saturated NaCl solution overnight under dark conditions. The slides were then treated at 50 ° C. for 15 minutes and blocked unreacted surface areas with 3D Link® blocking buffer. The slides were washed twice with water, then at 50 ° C. for 30 minutes with 0.2% SDS, finally washed twice with water and then air dried. Fluorescence detection was performed for a 20 second integration time on a Pharmacis scanner. Image and intensity profiles are shown in FIG. 25.
[200] Nonspecific oligonucleotides generated signals at 10 × 10 ³ to 25 × 10 ³ relative units at 10 μM. Signal intensity was compared to that observed for oligonucleotides containing a single amino group. In contrast, hydrazide modified oligonucleotides provided much more loading of 35-40 × 10 ³ fluorescent units. In addition, the hydrazide modified oligonucleotides showed a high fluorescence signal at low concentrations and a lower detection limit of 1.25 μM compared to the amine modified oligomer having a lower detection limit of 5 μM.
[201] Experimental Example 8.2: Attachment to Superaldehyde Slide
[202] Oligonucleotides were dissolved in Thermodix 3D Link® Print Buffer at pH 8.5, or 10 mM ammonium acetate buffer at pH 4.0, with concentrations ranging from 10 μΜ to 100 nM. From each solution, 0.5 μM was added to the surface of a superaldehyde slide (Telechem International, Inc, Sunnyvale, Calif.) And incubated overnight at room temperature. The slides were then treated twice with 0.2% SDS and washed four times with water (2 minutes each). The surface was then treated with a solution of 0.3% NaBH ₃ CN in PBS buffer (pH = 7) and bubbling was removed with 133 mL of ethanol. Washes were performed three times for one minute with 0.2% SDS and water. Fluorescence detection was performed for 20 seconds integration time with a Pharmacy scanner. Image and intensity profiles are shown in FIG. 26.
[203] As can be seen in FIG. 26, the hydrazide oligonucleotides at both pH 8.5 and 4.0 provided much higher signal strength compared to amine terminated oligomers and were not affected by changes in pH. Also, at the same concentration, hydrazide modified oligomers provided much higher signal strength than amine modified oligomers. Amine oligonucleotides were no longer detected at less than 2.5 μM, while hydrazide oligomers were detected as low as 1.25 μM.
[204] The foregoing is intended to provide examples of embodiments of the invention and is not intended to limit the invention in any way. While the present invention has been described with respect to particular variations, it should not be considered that the details thereof are limited, and that various modifications, changes, and variations may be made without departing from the spirit and scope of the invention, and such equivalent embodiments may be It is contemplated to be included herein. All publications and patent documents are included herein to the same extent as if each individual document or patent application was specifically and individually indicated to be incorporated by reference.

权利要求:
Claims (36)
[1" claim-type="Currently amended] (a) providing a biomolecule,
(b) contacting the biomolecule with a branched linking moiety to form a branched linking structure capable of coupling with a binding moiety on a substrate, and
(c) contacting the linking structure with a binding moiety included in the substrate to form a coupled substrate binding structure to which the biomolecules are bound.
Comprising, the biomolecules to the substrate.
[2" claim-type="Currently amended] The method of claim 1, wherein the branched connection structure is activated to form a coupled substrate bonding structure.
[3" claim-type="Currently amended] The method of claim 1 wherein the substrate is a polymer.
[4" claim-type="Currently amended] The method of claim 1 wherein the substrate is an activated polymer.
[5" claim-type="Currently amended] The method of claim 3 or 4, wherein the substrate is contacted with an electronically addressable microchip.
[6" claim-type="Currently amended] The method of claim 3 or 4, wherein the substrate is an activated glass slide.
[7" claim-type="Currently amended] The method of claim 3 or 4, wherein the substrate is a glass slide.
[8" claim-type="Currently amended] The method of claim 1, wherein the linking structure of the biomolecule comprises a Lewis base or nucleophile.
[9" claim-type="Currently amended] The method of claim 1, wherein the linking structure of the biomolecule comprises a Lewis acid or an electrophile.
[10" claim-type="Currently amended] The method of claim 8, wherein the Lewis base or nucleophile is selected from the group consisting of alcohols, amines, hydrazines, hydrazides, salicylic hydroxamic acid and sulfhydryls.
[11" claim-type="Currently amended] The method of claim 9 wherein the Lewis acid or electrophile is epoxide, aziridine, vinyl, aldehyde, ketone, acetal, disulfide, carboxylic acid, amide, bromo or iodoacetamide, N-hydroxysuccinimidyl Ester, sulfo-N-hydroxysuccinimidyl ester, azlactone, isocyanate, thioisocyanate, phenyl boronic acid and carbonate.
[12" claim-type="Currently amended] The method of claim 1, wherein the binding moiety of the biomolecule comprises a Lewis acid or an electrophile.
[13" claim-type="Currently amended] The method of claim 1, wherein the binding moiety comprises a Lewis base or nucleophile.
[14" claim-type="Currently amended] The method of claim 12 wherein the Lewis acid or electrophile is epoxide, aziridine, vinyl, aldehyde, ketone, acetal, disulfide, carboxylic acid, amide, bromo or iodoacetamide, N-hydroxysuccinimidyl Ester, sulfo-N-hydroxysuccinimidyl ester, azlactone, isocyanate, thioisocyanate, phenyl boronic acid, phosphoramidite and carbonate.
[15" claim-type="Currently amended] The method of claim 13, wherein the Lewis base or nucleophile is selected from alcohols, amines, hydrazines, hydrazides, salicylic hydroxamic acid and sulfhydryls.
[16" claim-type="Currently amended] The method of claim 1 wherein said branched linking moiety is phosphoramidite.
[17" claim-type="Currently amended] 17. The method of claim 16, wherein said phosphoramidite is
Diethyl 3-[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] glutarate,
Diethyl 5-{[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] methyl} isophthalate,
Dimethyl 3,3 '-(2-{[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] methyl} -2-{[2- (methoxycarbonyl) ethoxy] methyl} propane -1,3-diylbisoxy) dipropionate,
Ethyl 6-[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] hexanoate, and
6-[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] -N'-tritylhexanohydrazide
The method is selected from the group consisting of.
[18" claim-type="Currently amended] The method of claim 16, wherein the branched linking moiety forms a branched linking structure that is activated to bind to a substrate.
[19" claim-type="Currently amended] The method of claim 1, wherein the biomolecule and the substrate are connected via covalent bonds.
[20" claim-type="Currently amended] The method of claim 1, wherein the biomolecule and the substrate are connected through non-covalent bonds.
[21" claim-type="Currently amended] (a) coupling the biomolecule to a phosphoramidite comprising a protected hydrazide or hydrazide precursor, and
(b) contacting the protected hydrazide or hydrazide precursor with one or more reagents to convert the protected or precursor form to a hydrazide moiety.
Comprising, hydrazide to the biomolecule.
[22" claim-type="Currently amended] The method of claim 21 wherein the phosphoramidite is
Diethyl 3-[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] glutarate,
Diethyl 5-{[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] methyl} isophthalate,
Dimethyl 3,3 '-(2-{[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] methyl} -2-{[2- (methoxycarbonyl) ethoxy] methyl} propane -1,3-diylbisoxy) dipropionate,
Ethyl 6-[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] hexanoate, and
6-[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] -N'-tritylhexanohydrazide
The method is selected from the group consisting of.
[23" claim-type="Currently amended] Diethyl 3-[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] glutarate,
Diethyl 5-{[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] methyl} isophthalate,
Dimethyl 3,3 '-(2-{[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] methyl} -2-{[2- (methoxycarbonyl) ethoxy] methyl} propane -1,3-diylbisoxy) dipropionate,
Ethyl 6-[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] hexanoate, and
6-[(2-cyanoethoxy) (diisopropylamino) phosphanyloxy] -N'-tritylhexanohydrazide
Composition comprising phosphoramidite selected from the group consisting of.
[24" claim-type="Currently amended] The method of claim 21, wherein said biomolecule is first activated and then attached to a substrate comprising a binding moiety selected from the group consisting of aldehydes, ketones, esters, activated esters, acetals, haloacetamides and alkyl halides.
[25" claim-type="Currently amended] The biomolecule of claim 24 together with the protected hydrazide or hydrazide precursor is first converted to hydrazide, and then the group consists of 2-formylphenylboronic acid, 3-formylphenylboronic acid and 4-formylphenylboronic acid. A method of activating the biomolecule, which is treated with a reagent selected from.
[26" claim-type="Currently amended] The method of claim 25, wherein the activated biomolecule is reduced.
[27" claim-type="Currently amended] (a) coupling an oligonucleotide to a hydrazide moiety in the form of one or more precursors, and
(b) contacting the precursor form with one or more reagents to convert the precursor to a hydrazide residue
A method for producing a hydrazide-containing oligonucleotide comprising a.
[28" claim-type="Currently amended] The method of claim 27, wherein the hydrazide moiety in the form of the precursor is selected from the group consisting of aldehydes, ketones, esters, carboxylic acids, activated esters, acetals, haloacetamides, alkyl halides and trityl hydrazides. .
[29" claim-type="Currently amended] The method of claim 1,
(a) contacting the branched linking structure with a boronic acid containing moiety, and
(b) contacting the boronic acid containing moiety to the substrate
How to further include.
[30" claim-type="Currently amended] Use of branched and unbranched phenyl boronic acid containing molecules in binding biomolecules to a substrate surface comprising salicylic hydroxamic acid on an electronically addressable microchip.
[31" claim-type="Currently amended] Use of branched and unbranched hydrazide containing biomolecules that bind to a substrate surface comprising an active ester on an electronically addressable microchip.
[32" claim-type="Currently amended] The use according to claim 31, wherein the active ester is selected from the group consisting of azlactone, N-hydroxy succinimidyl ester and sulfo N-hydroxy succinimidyl ester.
[33" claim-type="Currently amended] Use of branched and unbranched hydrazide containing biomolecules that bind to a substrate surface comprising an aldehyde on an electronically addressable microchip.
[34" claim-type="Currently amended] Use of branched and unbranched hydrazide containing biomolecules that bind to a substrate surface comprising ketones on an electronically addressable microchip.
[35" claim-type="Currently amended] Wherein each biomolecule is bound to it by a plurality of binding moieties, the binding moiety being coupled to the biomolecule by a branched linking structure.
[36" claim-type="Currently amended] 36. The method of claim 35, wherein the microarray has a density of bound biomolecules in excess of the array 103 biomolecules per ² ㎛ dog.

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同族专利:

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公开号 | 公开日

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EP1257695A1|2002-11-20|

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WO2001051689A1|2001-07-19|

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DE60033851D1|2007-04-19|

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US7186813B1|2007-03-06|

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DE60033851T2|2007-11-08|

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EP1257695B1|2007-03-07|

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JP4731781B2|2011-07-27|

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AU6769700A|2001-07-24|

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JP2003521680A|2003-07-15|

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AT356231T|2007-03-15|

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CA2397091A1|2001-07-19|

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EP1257695A4|2005-03-02|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2000-01-11|Priority to US17555000P

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2000-01-11|Priority to US60/175,550

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2000-08-11|Application filed by 나노겐 인코포레이티드

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2000-08-11|Priority to PCT/US2000/022205

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2002-08-13|Publication of KR20020065644A

优先权:

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申请号 | 申请日 | 专利标题

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US17555000P| true| 2000-01-11|2000-01-11|

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US60/175,550|2000-01-11|

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PCT/US2000/022205|WO2001051689A1|2000-01-11|2000-08-11|Biomolecules having multiple attachment moieties for binding to a substrate surface|