WO2013040007A2

WO2013040007A2 - Devices and methods relating to immobilized ligands

Info

Publication number: WO2013040007A2
Application number: PCT/US2012/054816
Authority: WO
Inventors: Wei Shen
Original assignee: Regents Of The University Of Minnesota
Priority date: 2011-09-12
Filing date: 2012-09-12
Publication date: 2013-03-21
Also published as: WO2013040007A3

Abstract

In one aspect, this disclosure describes a device that generally includes a substrate and a ligand immobilized to the substrate through a compound comprising a heterodimerizable domain. In another aspect, this disclosure describes a method for detecting and/or separating a component of a biological sample. The method generally includes providing a device comprises a substrate and a ligand immobilized to the substrate via a polypeptide comprising a first heterodimerizable domain; contacting a biological sample with the device, the sample comprising at least component that selectively binds to the ligand; and detecting the at least one component of the sample bound to the ligand. In some embodiments, the binding of a component of the sample to the ligand may be dynamically and reversibly controlled under physiological conditions by controlling accessibility of the ligand. This may be accomplished by providing a blocking compound that generally includes a blocking moiety and a second heterodimerizable domain that can reversibly bind to the first heterodimerizable domain.

Description

DEVICES AND METHODS RELATING TO IMMOBILIZED LIGANDS

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Patent Application Serial No. 61/533,441, filed September 12, 2011, which is incorporated herein by reference in its entirety.

SUMMARY OF THE INVENTION

In one aspect, this disclosure describes a device that generally includes a substrate and a ligand immobilized to the substrate through a compound comprising a heterodimerizable domain. In some cases, the device can further include a blocking compound comprising a second heterodimerizable domain, wherein the second heterodimerizable domain is reversibly bound to the first heterodimerizable domain. In some embodiments, the first and second heterodimerizable domains can include coiled coil (e.g., leucine zipper) domains; in other embodiments, the first and second heterodimerizable domains can include generally complementary nucleotides sequences. In some embodiments, the device can further include a compound, molecular complex, cell fragment, or whole cell selectively bound to the ligand.

In another aspect, this disclosure describes a method for detecting and/or separating a component of a biological sample. The method generally includes providing a device that generally includes a substrate and a ligand immobilized to the substrate via a compound that includes a first heterodimerizable domain; contacting a biological sample with the device, in which the sample includes at least one component that selectively binds to the immobilized ligand; and detecting the at least one component of the sample bound to the ligand.

In some embodiments, detecting the at least one component can include separating the component bound to the ligand from at least one unbound component of the biological sample.

In some embodiments, the method includes providing a device that further includes a blocking compound comprising a second heterodimerizable domain, wherein the second heterodimerizable domain is reversibly bound to the first heterodimerizable domain, and contacting the device with a non-immobilized compound that includes the first

heterodimerizable domain, thereby disrupting the reversible binding between at least one first heterodimerizable domain and at least one second heterodimerizable domain.

In some embodiments, the first and second heterodimerizable domains can include leucine zipper domains; in other embodiments, the first and second heterodimerizable domains can include generally complementary nucleotides sequences.

In certain embodiments, the component of the sample that selectively binds to the ligand can include a compound, a molecular complex, a cell fragment, or a whole cell.

In certain embodiments, the method can further include perforrning an assay on at least a portion of the component of the sample bound to the ligand.

In certain embodiments, the method can further include releasing at least a portion of the component of the sample bound to the ligand.

In certain embodiments, the method can further include collecting at least a portion of the component of the sample bound to the ligand.

The above summary is not intended to describe each possible embodiment or every implementation of the present invention. The description that follows more particularly describes illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1. Illustration of the molecular design of bioactive surfaces capable of dynamically and reversibly regulating immobilized ligands (a) and the sequences of the polypeptides used in the study (b). Experimental polypeptides include cysARGD (SEQ ID NO:l), Beys (SEQ ID NO:2), and A (SEQ ID NO:3). Control polypeptides include cysA (SEQ ID NO:4) and cysC₁₀RGD (SEQ ID NO:5). Peptide domains included in the experimental and control polypeptides are abbreviated as follows: [6H] (SEQ ID NO:6), [A] (SEQ ID NO:7), [B] (SEQ ID NO:8), and [C₁₀] (SEQ ID NO:9).

FIG. 2. The shielding effect provided by co-immobilized PEG as revealed from a cell adhesion assay, (a) The cysARGD-functionalized surface, (b) The cysA-functionalized surface, (c-f) The cysARGD-functionalized surfaces further modified with B-PEG conjugates having PEG lengths of 10 kDa (c), 3.4 kDa (d), 700 Da (e), and 258 Da (f), respectively. The scale bars are 200 μηι.

FIG. 3. Reversible switch of the accessibility of immobilized RGD to cell surface receptors under physiological conditions, (a) The cells adhered to the cysARGD- functionalized surface as shown in FIG. 2a detached when B-PEG (PEG: 10 kDa) was added in the culture medium, (b) The cells adhered to a cysCioRGD-functionalized control surface did not detach when B-PEG (PEG: 10 kDa) was added, (c) The non-adhesive surface as shown in FIG. 2c became cell-adhesive when non-immobilized polypeptide A was added in the culture medium. The scale bars are 200 μπι.

FIG. 4. Gel electrophoresis data, (a) SDS-PAGE of cysARGD (lane 1), Beys (lane 2), and B-PEG (PEG: 10 kDa) (lane 3) confirmed that protein expression, protein purification, and the conjugation between Beys and PEGDA were successful, (b) Native gel

electrophoresis of cysARGD (lane 1), B-PEG (PEG: 10 kDa) (lane 2), and their mixture (lane 3) revealed a mobility shift of cysARGD after incubation with the B-PEG, suggesting that B retained its structure and function after conjugation. Slow migration of the molecules containing the B domain during native gel electrophoresis resulted from its molecular characteristics.

FIG.5. Illustration of using complementary DNA strands to design molecular switches and materials for cell isolation and non-enzymatic release. Molecules shown include DNA-1 (SEQ ID NO:10), DNA-2 (SEQ ID NO:l 1), and a peptide ligand (SEQ ID NO:13).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

To mimic the dynamic regulation of signaling ligands immobilized on extracellular matrices or on the surfaces of neighboring cells for guidance of cell behavior and fate selection, we have harnessed biomolecular recognition in combination with polymer engineering to create dynamic surfaces on which the accessibility of immobilized ligands to cell surface receptors can be reversibly interconverted under physiological conditions.

The dynamic surfaces exploit the reversible, noncovalent interaction between a first heterodimerizable domain that tethers a bioactive ligand to a substrate and a second heterodimerizable domain capable of dimerizing with the first heterodimerizable domain that is attached to a blocking compound. When the heterodimer is fomed, the blocking compound can shield the bioactive ligand so that the bioactive compound is generally non-accessible to components of the sample that is subject to assay and/or processing. Because the interaction between the first heterodimerizable domain and the second heterodimerizable domain in reversible and noncovalent, the bioactive ligand can be converted from the non-accessible state to an accessible state non-enzymatically by simply adding non-immobilized compounds that include the first heterodimerizable domain. The first heterodimerizable domain on these non-immobilized compounds can compete with the immobilized first heterodimerizable domains for binding to the second heterodimerizable domains. As the non-immobilized first heterodimerizable domains bind to the second heterodimerizable domains, the blocking compounds are removed from the immobilized first heterodimerizable domains, which consequently removes the shielding effect of the blocking compound and leaves the bioactive ligand accessible to components of the sample.

Sample components captured by bioactive ligands in the accessible state may be subjected to analysis and/or subsequently released from the ligand non-enzymatically. Thus, the platform described herein can be used as an assay platform or may be used for sample processing.

We demonstrated the functionality of the platform using the cell-adhesive RGD peptide as a model ligand. In this exemplary embodiment, RGD is fused to the C-terminus of a leucine zipper domain A and this fusion polypeptide is immobilized on the surface of a substrate, directly or indirectly, through a residue at the N-terminus of the leucine zipper domain (FIG. 1(a), left). The immobilized RGD can be converted from a cell-accessible to a cell-inaccessible state by adding a blocking compound— in this exemplary embodiment, a conjugate of poly(ethylene) glycol (PEG) and another leucine zipper domain B (B-PEG). Heterodimerization between A and B allows immobilization of at least some of the blocking compound (referred to herein as "co-immobilization") of the PEG, which can shield the RGD ligand from access by cells (FIG. 1(a), center). The shielded ligand can be converted back to a cell-accessible state by adding non-immobilized polypeptide A, which competes with the immobilized A for binding to B-PEG and decreases the extent of co-immobilization, thereby decreasing the extent to which the PEG blocking compound shields the RGD ligand. This molecular design can offer one or more benefits including, for example, the interconversion is reversible, the ligand can remain immobilized during dynamic regulation so that cells are not exposed to the soluble form of the ligand that potentially has detrimental effects, and the precision of the on/off states can be maintained by the molecular-level uniformity of the ligand and PEG co-immobilized through leucine zipper heterodimerization. The method can be readily adapted for dynamic regulation of other immobilized bioactive ligands of interest. This platform therefore provides a convenient way to assay and/or process a particular component of a complex mixture.

Using engineered materials to recapitulate the essential spatial and temporal characteristics of natural extracellular microenvironments during animal development is one challenge to successfully guiding cell behavior and fate selection in tissue engineering (Lutolf and Hubbell, Nat. Biotechnol. 2005, 23:47-55; Lee et al, Nature 2000, 408:998-1000; Kloxin et al, Science 2009, 324:59-63; Campbell et al., Biomaterials 2005, 26:6762-6770; DeLong et al, Biomaterials 2005, 26:3227-3234; DeForest et al., Nat. Mater. 2009, 8:659-664). One aspect of this challenge involves mimicking the dynamic regulation of signaling ligands immobilized on extracellular matrices or on the surfaces of neighboring cells. Although soluble ligands can be added or removed readily during in vitro studies, the soluble form of a ligand that performs its function in an immobilized state in vivo potentially has opposite and detrimental effects because of competitive binding for cell surface receptors. This has been demonstrated by inhibition of Notch signaling in the presence of non-immobilized Notch ligands and death of anchorage-dependent cells in the presence of soluble RGD peptide (V as et al., Leukoc. Biol. 2004, 75:714-720; Varnum-Finney et al., J Cell Sci. 2000, 113:4313- 4318; Michel, Arterioscler. Thromb. Vase. Biol. 2003, 23:2146-2154). In recent years, materials capable of dynamically presenting immobilized bioactive ligands to cell surface receptors under physiological conditions have attracted increasing research interest (Salinas and Anseth, Biomaterials 2008, 29:2370-2377; Yeo et al, J Am. Chem. Soc. 2003,

125:14994-14995; Chan et al., Angew. Chem., Int. Ed. 2008, 47:6267-6271; Todd et al., Soft Matter 2007, 3:547-550; Petersen et al., Angew. Chem., Int. Ed. 2008, 47:3192-3195;

Auernheimer et al, J Am.Chem. Soc. 2005, 127:16107-16110). However, the methods developed to date have drawbacks such as, for example, irreversible or imprecise transition between the on/off states and/or exposure of cells to the soluble counterparts of the ligands during in situ immobilization or cleavage. The limited ability to dynamically regulate immobilized bioactive ligands in engineered systems has hindered the progress of both technological development and fundamental understanding in tissue engineering.

Here we report a novel method of harnessing the molecular recognition of

biomolecules in combination with polymer engineering to create dynamic bioactive surfaces on which the accessibility of immobilized ligands to cell surface receptors can be reversibly interconverted with high precision under physiological conditions. The accessibility of an immobilized bioactive ligand to cells can be dynamically regulated through reversible co- immobilization and removal of a blocking compound such as, for example, poly(ethylene) glycol (PEG) that, when co-immobilized, shields the surface-bound ligand from access by cells, cell fragments, compounds, or molecular complexes.

Thus, reversible co-immobilization and removal of the blocking compound can be achieved by exploiting the molecular recognition between a pair of complementary heterodimerizable compounds— e.g., coiled coil polypeptide domains or polynucleotide sequences— that heterodimerize through noncovalent, reversible interactions.

In one exemplary embodiment, illustrated in FIG. 1(a), a bioactive ligand of interest is fused to one terminus of the leucine zipper A and immobilized on a surface, directly or indirectly, through a residue at the other terminus. The modified surface allows co- immobilization of a leucine zipper B-blocking compound conjugate, which can shield the surface-bound bioactive ligand and can convert it from an accessible state to an inaccessible state. To convert the ligand back to a accessible state, non-immobilized polypeptide A can be added in excess to compete with the immobilized A for binding to the B-blocking compound conjugate, thereby decreasing the quantity of B-blocking compound conjugate bound to immobilized A and in place to shield the ligand. This molecular design allows the

immobilized ligand to be reversibly interconverted between accessible and inaccessible states under physiological conditions as many times as needed. The bioactive ligand remains immobilized during the dynamic regulation, so cells (or other target components in the sample) are less likely to be exposed to the soluble counterpart of the ligand.

One feature of this design is that heterodimerization between, for example, A and B, allows the bioactive ligand and blocking compound to be co-immobilized uniformly on the molecular level, which can limit— and in some cases even prevent— the development of ligand islands that are free of the blocking compound. Such islands otherwise often form when, for example, a conventional co-immobilization method is used. Moreover, such islands can compromise the precise switch of the ligand between cell-accessible and cell-inaccessible states.

Polypeptides and their amino acid sequences of one exemplary embodiment are shown in FIG. 1(b). The complementary leucine zipper domains A and B were used because they could heterodimerize with an affinity (in the range between 10^"8 M and 10^"10 M) higher than that of homo-oligomerization (self-aggregation) of A or B (Shen et al., Nat. Mater. 2006, 5:153-158; McGrath et al., J Bioact. Compat. Pofym. 2000, 15:334-356). The cell-adhesive RGD peptide was chosen as a model bioactive ligand. A cysteine residue was engineered at the N-terminus of A for surface immobilization. All of the polypeptides were genetically engineered and biosynthesized. B-PEG conjugates were prepared by allowing Beys to react with a large excess of PEG-diacrylate through a Michael-type addition reaction or with a large excess of PEG-maleimide through the tMol-maleimide reaction. After conjugation with PEG, B retained its ability to heterodimerize with A, as revealed from the mobility shift of cysARGD in native gel electrophoresis after cysARGD was incubated with B-PEG (FIG. 4).

The ability of the surface-immobilized cysARGD to co-immobilize B-PEG through heterodimerization between A and B and the ability of the co-immobilized PEG to shield the surface-bound RGD from access by cells were examined. First, cysARGD and a control polypeptide cysA were each immobilized on piranha-solution-cleaned gold surfaces through the cysteine residue in the presence of 5 rnM s(2-carboxyethyl)phosphine hydrochloride (TCEP). The surfaces modified with cysARGD supported the adhesion of fibroblasts (NIH 3T3) while those modified with cysA did not (FIG. 2(a) and FIG. 2(b)), suggesting that cell adhesion on cysARGD-functionalized surfaces was regulated through the specific interaction between the RGD peptide and cell surface receptors. Then the cysARGD-functionalized surfaces were incubated with B-PEG conjugates of various PEG lengths (10 kDa, 3.4 kDa, 700 Da, and 258 Da), resulting in further modified surfaces exhibiting different cell adhesion properties depending on the PEG size. When the PEG was 10 kDa, the surfaces did not support cell adhesion (FIG. 2(c)); when the PEG was 3.4 kDa, the surfaces partially supported cell adhesion (FIG. 2(d)); and when the PEG was 700 Da or smaller, the surfaces were as adhesive as the cysARGD-functionalized surfaces without additional modification (FIG. 2(e) and FIG. 2(f)). These results suggested that the immobilized cysARGD allowed co- immobilization of B-PEG through heterodimerization between A and B and the shielding effect provided by the co-immobilized PEG increased with its size.

The reversible switch of the surface-bound RGD between cell-accessible and cell- inaccessible states was performed in the presence of cells under physiological conditions. To demonstrate the accessible-to-inaccessible conversion of the immobilized RGD peptide, fibroblasts were allowed to adhere on a surface as shown in FIG. 2(a) for three hours. Then B- PEG (PEG: 10 kDa, 300 μΜ) was added into the culture medium, followed by incubation under shaking (at a speed at which the liquid just started to move to enhance mass transfer) for two hours. Cell detachment was observed (FIG. 3(a)). A control performed on a cysC₁₀RGD-functionalized surface (C₁₀ is a hydrophilic random coil; Shen et al., Nat. Mater. 2006, 5:153-158) under the same condition did not result in cell detachment (FIG. 3(b)), suggesting that the cell detachment from the cysARGD-functionalized surface was caused by co-immobilization of B-PEG through heterodimerization between A and B. These results not only suggested that the surface-bound cysARGD allowed co-immobilization of B-PEG in the presence of cells under physiological conditions but also showed that even the ligands that had akeady been engaged in the interactions with cell surface receptors could be converted to the cell-inaccessible state due to the physical, reversible, and dynamic nature of the interactions between ligands and cell surface receptors.

To demonstrate that the cell-inaccessible RGD shielded by PEG could be converted back to the cell-accessible state in the presence of cells, a cysARGD-functionalized surface was further modified with B-PEG (PEG: 10 kDa); cells were seeded on the resulting non- adhesive surface. Non-immobilized polypeptide A (300 μΜ) was added in the cell culture medium, followed by shaking incubation (to enhance mass transfer) for two hours and static incubation (to allow cell adhesion) for three hours sequentially. The surface was converted from non-adhesive (FIG. 2(c)) to cell-adhesive (FIG. 3(c)), suggesting that the non- immobilized A competed with the immobilized cysARGD for binding with B-PEG and most B-PEG molecules could be removed from the surface when the amount of non-immobilized A was sufficiently greater than that of immobilized cysARGD. The RGD peptide in

immobilized cysARGD was consequently converted from the cell-inaccessible to cell- accessible state. After the medium was changed, the cell-accessible RGD could be reconverted to be cell-inaccessible by addition of B-PEG.

While described above with respect to an embodiment in which the ligand— the RGD peptide— is selected to selectively bind to a surface receptor of a whole cell, the platform may be equally useful for the selective, dynamic, reversible, affinity-based binding of individual compounds, molecular complexes, or cell fragments. Consequently, the ligand may be any appropriate ligand for the selective affinity-based capture of any particular target. Exemplary ligands include, for example: cell adhesion peptide ligand such as, for example, peptides that interact with integrins (e.g., RGD, which binds to multiple integrins, REDV, which binds to α4β1 integrin, and DGEA, which binds to α2β1 mtegrin), peptides that interact with cadherins (e.g., HAV, which binds to N-cadherin), and peptides that interact with other cell surface proteins (e.g., IKVAV, which binds to LBPl 1); peptide ligands that interact with cell surface receptors (e.g., GGKRPAR, which binds to neuropilin-1); antibodies or fragments thereof— e.g., single-chain antibodies— that selectively bind to cell surface receptors (e.g., CD34) or to compounds or molecular complexes of interest; and/or nucleic acid (DNA and/or RNA) aptamers that selectively bind to cell surface molecules (e.g., aptamers that selectively bind to surface molecules of sperm cells).

As used herein, binding is considered "selective" if the ligand has a differential or non-general affinity, to any degree, for a particular target. Thus, a ligand need not exhibit exclusive specificity for a particular target in order to "selectively" bind the target.

Also, while the device is described above in the context of having a single bioactive ligand, a device may be designed with a plurality of ligands if one desires to simultaneously capture more than one component of a complex mixture being assayed and/or processed. When a plurality of ligands is used, the various ligands may be arranged in any suitable manner such as, for example, dispersed uniformly over the surface of the substrate, localized in discrete, addressable locations on the substrate, or in any other desirable manner.

In addition, while the platform is described above in the context of leucine zipper domains as the heterodimer, the accessibility and inaccessibility of the capturing ligand can be dynamically controlled exploiting the reversible, noncovalent binding of any

heterodimerizable pair such as, for example, other coiled coil polypeptide heterodimers, generally complementary nucleic acid molecules, and protein-small molecule pairs such as, for example, streptavidin and biotin.

Suitable heterodimerizable pairs include any pair of heterodimerizable molecules capable of reversible, noncovalent self-assembly at an appropriate temperature for the assay being performed. In many embodiments, the heterodimerizable pair can include biopolymer compounds such as, for example, polypeptides or nucleic acid molecules. In other

embodiments, however, the heterodimerizable pair may not necessary require a biopolymer. Typically, appropriate temperatures reflect physiological temperatures and therefore are often in the range of from about 25°C to about 45°C, although under certain circumstances the platform may be used to perform an assay outside of this range. As used herein, the term "self-assembly" refers generically to the property of two heterodimerizable molecules to reversibly assemble noncovalently into a dimeric complex. In the context of nucleic acid molecules, the term "self-assembly" is more typically referred to as hybridization.

As used herein, the term "generally complementary nucleic acid molecules" refers to nucleic acid molecules that are generally complementary without regard to whether either molecules is RNA, DNA, or an artificially-created nucleic acid molecule. The nucleic acid molecules need not be 100% complementary in order to provide the desired controlled reversible binding in physiological conditions. Thus, the 100% complementary nucleotide molecules SEQ ED NO: 10 and SEQ ID NO:l 1 shown in FIG. 5(a) are merely exemplary. The platform described herein may use any pair of generally complementary nucleic acid molecules. Nucleic acid molecules are generally complementary if they are capable of "self- assembly" as described in the immediately preceding paragraph— i.e., if they are capable of hybridization at a temperature appropriate for the assay being performed under otherwise standard hybridization conditions.

In some embodiments, the heterodimerizable pair may be capable of self-assembly at a minimum temperature of at least 25°C such as, for example, at least 27°C, at least 30°C, at least 35°C, at least 37°C, or at least 40°C. In some embodiments, the heterodimerizable pah- may be capable of self-assembly at a maximum temperature of no more than 45°C such as, for example, no more than 40°C, no more than 37°C, or no more than 30°C. In some embodiments, the heterodimerizable pair may be capable of self-assembly within a range of temperatures having endpoints defined by any minimum temperature listed immediately above and any appropriate maximum temperature listed above.

Similarly, while described with respect to an exemplary embodiment in which the ligand is immobilized to a gold substrate via a cysteine residue at the N-terrriinal of the leucine zipper protein A, the platform is not limited by the substrate or the manner in which the heterodimerizable molecules is immobilized to the substrate. For example, a thiol- containing heterodimerizable molecule can be immobilized to the surface of a hydrogel functionalized with maleimide or ene (Kosif et al., Macromolecules 2010, 43:4140-4148; Aimetti et al., Biomaterials 2009, 30:6048-6054). Other techniques for immobilizing polypeptides, nucleic acids, and other heterodimerizable compounds (e.g., streptavidin and/or biotin) to a substrate are well known to those of ordinary skill in the art.

Also, while described in the context of an exemplary embodiment in which the blocking compound includes PEG, the platform described herein can include any suitable blocking compound. Suitable blocking compounds generally can include compounds that are soluble in aqueous solutions, can adopt a steric conformation that shields the bioactive ligand from components of the sample being assayed or processed, and are substantially inert to the components of interest in the sample being assayed and/or processed. Thus, a blocking compound may interact with components of the sample that are not the subject of the assay and/or processing. For example, if the sample component of interest is a specific cell type, it is no concern if the blocking compound interacts with, for example, serum antibodies or other cell types that may be present in the sample.

In some cases, the blocking compound can include, for example, a polymer capable of adopting an extended chain conformation. Such blocking compounds include, for example, organic polymers such as PEG and poly(vinyl alcohol). Other blocking compounds can include, for example, polypeptide-based polymer such as, for example, elastin-like polypeptides that have a transition temperature greater than the temperature at which the sample is being assayed and/or processed. Other suitable blocking compounds include polysaccharides such as, for example, agarose and alginate.

In some embodiments, the blocking compound may naturally include the second heterodimerizable domain. In other embodiments, the blocking compound may be engineered to include a heterologous domain that possesses the second heterodimerizable domain. Such engineered compounds can be produced using conventional recombinant, synthetic, and/or enzymatic techniques.

The method described herein will find use in many applications such as, for example, regulating stem cell differentiation, which is stepwise and often involved with changes in microenvironmental signals on the order of hours or days. While shaking incubation can improve the mass transfer and allow the interconversion of sites between the accessible and inaccessible state to occur at one hour or two hours (the experiment described in FIG. 3(a) was also tested at one hour and the same result was obtained), exposing cells to the shear stress associated with the convective flow may introduce some complexity. The fluid velocity required for convective mass transfer to dominate over diffusive mass transfer is small, however. For example, scaling analysis based on the length scale of cells and the time scale required for the interconversion to occur under static conditions— approximately 10 hours— suggests that a velocity on the order of 10^"6 cm/sec is enough to result in a Peclet number of at least about 100 (Deen, Analysis of Transport Phenomena; Oxford University Press, New York, 1998). Peclet number refers to the ratio of convective mass transfer to diffusive mass transfer. The shear stress associated with such small velocity is low.

In addition, this method potentially allows both temporal and spatial control of ligands. For example, a step gradient of a ligand can be created via micro/nano-printing, even though creation of a continuous gradient might be difficult.

In addition, methods exploiting the platform described herein can allow one to selectively capture cells, cell fragments, protein complexes, and/or compounds of interest from a complex mixture, then subsequently release the capture components non- enzymatically.

Another feature of the method is that the interconversion between the accessible state and the inaccessible state may not be instant.

In summary, the molecular engineering approach reported here represents a platform that can be readily and generally adapted to dynamically and reversibly regulate the accessibility of a variety of immobilized bioactive ligands to various targeted moieties under physiological conditions. The materials having such dynamic bioactive surfaces can be used to temporally control the properties of engineered extracellular microenvironments. The platform may be exploited in processes for, for example, separating the targeted moiety from a sample that contains a mixture of one or more additional components.

As used herein, the term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements; the terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims; unless otherwise specified, "a," "an," "the," and "at least one" are used

interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

In the preceding description, particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.

For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLES

Example 1

Synthesis and purification of polypeptides.

The genes encoding the polypeptides shown in FIG. 1(b) were each constructed in the Qiagen (V alencia, CA) pQE9 expression vector through standard recombinant DNA mampulation. Polypeptides were expressed under control of the bacteriophage T5 promoter in the E.coli strain SG13009 and purified through nickel-nitrilotriacetic acid (Ni-NTA) metal- affinity chromatography. The biosynthesized polypeptides were characterized using MALDI mass spectrometry and SDS-polyacrylamide gel electrophoresis (FIG. 4). Preparation of B-PEG conjugates.

B-PEG conjugates were prepared by allowing Beys to react with a large excess of PEG-diacrylate (PEGDA) or PEG-maleimide (Lutolf et al, Bioconjug. Chem. 2001, 12:1051- 1056; Liu et al., Biomacromolecules 2009, 10:3182-3187; Shen et al., Sofi Matter 2007, 3 :99- 107). Even though each PEGDA has two terminal acrylate groups, its large excess in the conjugation reaction prevents modification of both acrylate groups as revealed from MALDI mass spectrometric analysis. In other embodiments, conjugates between Beys and

monofunctional PEG-acrylate can also be used. For either bifunctional or monofunctional PEG, conjugation reactions were performed with a large excess of PEG to drive the modification of Beys to completion. One can minimize the amount of unmodified Beys to limit the extent to unmodified Beys competes with B-PEG for binding to immobilized cysA GD.

The data reported in FIG. 2 and FIG. 3 were all acquired with Bcys-PEGDA conjugates. The experiment described in FIG. 3(a) was repeated with the conjugate between Beys and PEG-maleimide, and the same result was obtained. Even though the ester bond formed between thiol and acrylate is hydrolysable with a half-life of approximately 11 days at the physiological pH, hydrolysis is negligible on the time scale of our experiments (Elbert and Hubbell, Biomacromolecules, 2001, 2:430-441; Schoenmakers et al., J Control Release 2004, 95:291-300). Future studies with longer time scales can be performed by using conjugates between Beys and PEG-maleimide.

PEGDA and PEG-maleimide were purchased from Laysan Bio (Arab, AL) and Sigma-Aldrich (St. Louis, MO). For conjugation, a Beys solution prepared in PBS was reduced by 30 raM tris(2-carboxyethyl)phosphine hydrochloride (TCEP HC1, Pierce) at pH 5.0 at room temperature for one hour, followed by removal of TCEP through a Microcon centrifugal filter unit (Millipore; Billerica, MA). The solution pH was adjusted to a desired value (8.0 for conjugation with PEGDA and 6.5 for conjugation with PEG-maleimide) and the two reactants were mixed at a 10:1 molar ratio (PEG: Beys). The mixture was stirred in the dark at room temperature for 12 hours, followed by removal of unreacted PEG through Ni- NTA metal-affinity chromatography. The B-PEG conjugates were characterized using MALDI mass spectrometry and SDS-polyacrylamide gel electrophoresis. To examine whether the B domain in B-PEG retained its structure and ability to heterodimerize with the A domain, a mixture of 100 μΜ cysARGD and 200 μΜ B-PEG (the conjugate of Beys with 10 kDa PEGDA) was prepared in PBS (pH 7.4) and incubated at room temperature for two hours. Native electrophoresis of cysARGD, B-PEG, and their mixture was conducted to reveal whether the mobility of cysARGD shifts after incubation with B-PEG (FIG. 4).

Immobilization of thiol-containing polypeptides.

Gold slides (EMF Inc.; Ithaca, NY) were cleaned in freshly prepared piranha solution (70% H₂S0₄, 30% H₂0₂) for two hours, followed by thorough washing with deionized water and PBS (Uzarski et al., Colloids Surf. B 2008, 67: 157-165). The solution of each thiol- containing polypeptide (cysARGD, cysA, or cysC₁₀RGD) was prepared at a concentration of 100 μΜ in PBS (pH 7.4) supplemented with 5 mM TCEP. Each cleaned slide was incubated in the solution of a tMol-containing polypeptide for six hours. The slide was washed with PBS, followed by cell adhesion assay or further modification with B-PEG.

Co-immobilization of B-PEG and examination of its shielding effect.

Solutions of B-PEG conjugates of various PEG lengths (10 kDa, 3.4 kDa, 700 Da, and 258 Da) were each prepared in PBS at a concentration of 300 μΜ. The cysARGD- functionalized surface was incubated with a B-PEG solution overnight, followed by washing with PBS and serum-free Dulbecco's Modified Eagle Medium (DMEM) (5 times for each). Fibroblasts (NIH 3T3) were seeded on each surface at a density of 80,000 cells/cm² in serum- free DMEM and incubated in 5% C0₂ at 37°C for three hours to allow adhesion. For the cell adhesion assay, each sample was washed to remove non-adherent cells; adherent cells were stained with the calcein AM dye. The sample was placed in a tissue culture well with the side having cells facing down and examined with a 10 objective on a Zeiss Axio Observer inverted fluorescent microscope.

Reversible switch of ligand accessibility for cells.

To demonstrate that the immobilized RGD in the surface-bound cysARGD could be switched from the cell-accessible to cell-inaccessible state in the presence of cells under physiological conditions, cysARGD and a control polypeptide cysC₁₀RGD were each immobilized on gold surfaces. Fibroblasts were seeded in serum-free DMEM at a density of 80,000 cells/cm² and incubated for three hours to allow cell adhesion. B-PEG (PEG: 10 kDa) was added in the medium of each sample at a concentration of 300 μΜ and incubation was continued for an additional two hours under shaking (at a speed at which the liquid just started to move), followed by the cell adhesion assay.

To demonstrate that cell-inaccessible RGD shielded by co-immobilized PEG could be switched to the cell-accessible state in the presence of cells under physiological conditions, cysARGD-functionalized surfaces with co-immobilized B-PEG (PEG: 10 kDa) were prepared. Fibroblasts were seeded on these non-adhesive surfaces at a density of 80,000 cells/cm² in serum-free DMEM. A solution of non-immobilized polypeptide A was added into the cell culture medium at a final concentration of 300 μΜ, followed by incubation under shaking for two hours, static incubation for three hours, and the cell adhesion assay. All the incubation steps were performed in a tissue culture incubator (5% C0₂, 37°C).

To confirm the interconversion is reversible, two cycles of switch have been performed on the same sample, with the initial state of the immobilized RGD being either cell-accessible or cell-inaccessible.

Example 2

Preparation and immobilization of a peptide-DNA conjugate

As one example, complementary DNA strands have the following sequences: 5'- tgagtgtaccaccattggaag-3' (DNA-1; SEQ ID NO:10) and 5'-cttccaatggtggtacactca-3' (DNA-2; SEQ ED NO:l 1). DNA-1 is conjugated to a peptide ligand REDV (SEQ ID NO: 12), which specifically interacts with the α4β1 integrin. The DNA-1 -peptide conjugate is immobilized on a substrate for cell capture. DNA-2 is conjugated to a PEG chain and the DNA-2-PEG conjugate serves as a releasing agent (FIG. 5).

5'-azide-modified and 3'-thiol-modified DNA-1 are commercially available

(Integrated DNA technologies; Coralville, LA). The 3' thiol is used for conjugation with the peptide. The 5' azide is used for surface immobilization. The peptide SGSGSGREDV (SEQ LD NO: 13) is prepared using solid-phase synthesis. To conjugate the peptide with DNA-1, the peptide is functionalized with a maleimide group by reacting with Sulfosuccinimidyl-4-(iV- maleimidomethyl)cyclohexane-l-carboxylate (Sulfo-SMCC, Thermo Scientific; Waltham, MA). The maleimide-functionalized peptide reacts with 3'-thiol-modified DNA-1 through the Mol-mdeimide reaction. The resulting conjugate carries a 5'-azide, which allows it to be immobilized on an alkyne-functionalized substrate through Cu-catalyzed azide alkyne cycloaddition to yield the affinity substrate.

Preparation of a releasing agent

5'-tbiol-modified DNA-2 is commercially available (Integrated DNA technologies; Coralville, IA). It conjugates with PEG-maleimide through the thiol-maleimide reaction to result in the releasing agent DNA-2-PEG.

Affinity capture of cells and non-enzymatic release of captured cells

Human umbilical vein endothelial cells (HUVECs) are labeled with CellTracker™ Green CMFDA (Invitrogen; Grand Island, NY); human aortic smooth muscle cells (AoSMCs) are labeled with CellTracker™ Orange CMTMR (Invitrogen; Grand Island, NY). HUVECs and AoSMCs are mixed and incubated on the substrate presenting immobilized DNA-1(SEQ ID NO: 10) - SGSGSGREDV (SEQ ID NO: 13) for three hours, followed by washing with PBS. Fluorescent microscopy reveals that HUVECs expressing the 4β1 integrin are selectively captured on the substrate. The captured cells are released after the releasing agent DNA-2-PEG is added. This non-enzymatic releasing method minimizes the perturbation of cell properties. The recovered cells exhibit normal viability, proliferative ability, and tube formation on Matrigel.

The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PHL, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

What is claimed is:

1. A device comprising:

a substrate; and

a ligand immobilized to the substrate by a compound comprising a heterodimerizable domain.

2. The device of claim 1 further comprising a blocking compound comprising a second heterodimerizable domain, wherein the second heterodimerizable domain is reversibly bound to the first heterodimerizable domain.

3. The device of either claim 1 or claim 2 wherein the first and second heterodimerizable domains comprise coiled coil polypeptide domains.

4. The device of either claim 1 or claim 2 wherein the first and second heterodimerizable domains comprise generally complementary nucleotides sequences.

5. The device of any preceding claim further comprising a compound, molecular complex, cell fragment, or whole cell selectively bound to the ligand.

6. A method comprising:

providing a device comprising:

a substrate; and

a ligand immobilized to the substrate by a compound comprising a first heterodimerizable domain;

contacting a biological sample with the device, the sample comprising at least one component that selectively binds to the ligand; and

detecting the at least one component of the sample bound to the ligand.

7. The method of claim 6 wherein the component comprises a compound, a molecular complex, a cell fragment, or a whole cell.

8. The method of claim 6 wherein detecting the at least one component comprises separating the component bound to the ligand from at least one unbound component of the biological sample.

9. The method of claim 6, wherein the device further comprises a blocking compound comprising a second heterodimerizable domain, wherein the second heterodimerizable domain is reversibly bound to the first heterodimerizable domain; and

contacting a biological sample with the device further comprises contacting the device with non-immobilized polypeptide comprising the first heterodimerizable domain, thereby disrupting the reversible binding between at least one first heterodimerizable domain and at least one second heterodimerizable domain.

10. The method of claim 9 wherein the first and second heterodimerizable domains comprise coiled coil polypeptide domains.

11. The method of claim 9 wherein the first and second heterodimerizable domains comprise generally complementary nucleotides sequences.

12. The method of any one of claims 6-11 wherein the component that selectively binds to the ligand comprises a compound, molecular complex, cell fragment, or whole cell.

13. The method of any one of claims 6-12 further comprising performing an assay on the at least one component of the sample bound to the ligand.

14. The method of any one of claims 6-13 further comprising releasing at least a portion of the component of the sample bound to the ligand.

15. The method of any one of claims 6-14 further comprising collecting at least a portion of the component of the sample bound to the ligand.