Display of molecules on genetically encoded silent nanoscale carriers for determining synergistic molecular interactions

Abstract

This application provides a method for generating "liquid" arrays of ligand- (e.g., glycan-) modified bacteriophages, where the ligand modifications are genetically encoded within the bacteriophage genome. This method allows for the determination of ligand-binding profiles of biopolymers and cells. Furthermore, this method allows for the elucidation of ligand-protein interactions where ligand binding is cooperative and synergistic.

Description

Technical Field

[0001] This application relates to the fields of receptor-ligand interactions and molecular recognition. More particularly, this application relates to methods for discovering ligands or combinations of ligands that bind synergistically to a target biomolecule.

Background Art

[0002] Many proteins and other macromolecular receptors are known to interact with multiple ligands. The simultaneous interaction of a receptor with two ligands often results in biophysical, biochemical, and physiological outcomes that are different from the interaction of the same receptor with either of the individual ligands. Such interactions are termed "synergistic" or "positively cooperative" when the binding of the two molecules is found to be more favorable than the binding of the individual ligands. 1 . These "synergistic" interactions can be a very large goal in the fields dealing with receptor-ligand interactions (drug discovery, diagnostics, basic research).

[0003] A specific example of a synergistic interaction is the interaction between carbohydrates and proteins. Examples are known where two different types of glycans bind to a single protein with a significantly higher affinity than either glycan alone. 2-4 . Among the possible factors, the biophysical origin of such enhancement could be due to allosteric conformational changes within the protein structure or the interaction of the two molecules.

[0004] Many known methods of ligand discovery are optimized for discovering individual ligands that bind to individual proteins, referred to herein as "spatially separated libraries". 2. Examples include screening a library of individual molecules on a microtiter plate, screening a molecular array where each molecule is attached to the surface at a specific position, or screening a one-bead-one-compound library where individual macroscopic (micron-sized) beads have unique molecules. To enable screening of synergistic interactions, it is theoretically possible to upgrade the "spatially separated library" technology, but in practice, it can become exponentially more complex. A library of N different molecules contains approximately N 2 / 2 unique binary combinations. Thus, even for a library of 1000 small molecules, it is necessary to create and test 500,000 binary combinations. For ternary combinations, this number becomes 200,000,000. Therefore, to achieve feasible results, it is necessary to reduce the complexity of the library (i.e., reduce the number of library members to be tested).

[0005] A well-known technique that complements the "spatially separated library" is the "mixed library" technique, where multiple molecules are present in the same solution. This technique enables screening of mixtures of molecules and is a "display" technique. In display techniques, each molecule covalently or non-covalently binds to a nanoscale information-containing tag such as DNA, RNA, ribosomes, or particles of bacteriophage or virus. A variant of such a technique is SELEX (systematic evolution of ligands by exponential enrichment) or a similar procedure for the development of RNA or DNA aptamers, where the encoded entity is a DNA or RNA molecule. However, DNA or RNA can potentially interact with receptors, and such interactions may or may not be desirable. These problems are minimized in phage display techniques where different molecules are immobilized on virus or bacteriophage particles of the same composition, and DNA or RNA of different compositions is contained within the viral capsid of the phage particle.

[0006] The mixed library technique is suitable for the identification of synergistic binding because all molecules are present in the same solution. However, the identification of synergistic interactions using mixed-coded libraries has not been documented. Several of the following requirements are not clear: (1) To analyze synergistic binding, it is necessary to be able to create a library of N defined components and a nearly identical library of N - m components with m specific members of the original library excluded (m < N). (2) In the generation and application of the mixed library technique, it is necessary to enable two or more molecules to interact with the same target. For example, Lerner and Brenner, Lam and co-workers, etc. have taught the creation of mixed molecular libraries displayed with coded tags on macroscopic carriers such as beads made of agarose, polystyrene, and larger than 1 micron in size. The size of the carrier beads effectively prevents different molecules attached to two different beads from simultaneously binding to one protein target with a size of less than 0.01 microns.

[0007] One technique for generating a display library on a nanoscale carrier of the same composition utilizes recombinant protein technology in which additional DNA is introduced into one gene of the phage coat protein, resulting in the production of protein fusion products packaged into virions or phage particles. Multiple variants of such phage and phagemid display technologies are known in the art and are designed to identify molecules that bind to a receptor of interest.

[0008] The display of phage M13 is a specific example of a genetically encoded library or "display technology". 5 Phage display is a well-known technique used for the analysis, display, and production of protein antigens, particularly human proteins of interest. 6。By genetic engineering of the M13 phage genome, the peptide or protein of interest binds individually to the phage virion surface protein molecule (usually the Gene III protein, g3p). In such a phage population (phage library), each phage has a gene for a different peptide or protein-g3p fusion exposed on its surface. Modification of the genome usually produces phage particles that are not chemically identical. These differences in chemical composition can contribute to differences in how these particles interact with the target. To mitigate this problem, silent coding can be used.

[0009] "Silent barcode" technology 7 has been described. This relates to a method of generating a bacteriophage display system on particles that contain DNA of different compositions within the bacteriophage particle and display peptides of the same composition. This technology enables facile chemical modification of existing peptide libraries with different chemical modifiers.

[0010] Various methods are known for tagging molecules with DNA or RNA. The tags of these technologies have different chemical compositions and can also form interactions with targets. The SELEX RNA and DNA aptamer technologies teach that different DNA or RNA sequences may have different degrees of interaction with biomolecules. 8,9 The results of screening using molecules tagged with DNA or RNA were not very predictable because there may be undesirable interactions between the target and the "tag".

[0011] Determining the glycan-binding profile of a lectin can be difficult and time-consuming. One current method for such identification often uses an array of glycans chemically conjugated to a solid surface, which is often glass. Using such a glycan array, the preference of a specific lectin for one or more specific glycans immobilized on the surface is determined using a two-step procedure. First, a large number of glycans are conjugated to one surface such that one glycan is present at each of a number of spatially distinct locations. The glycan array is then "panned" with a labeled biomolecule, and the preference of the biomolecule for the glycans is determined by detection of the label. The main advantage of this system is that the glycan-binding preference of a lectin or biomolecule for a large number of 50-200 glycans can be evaluated in a single format. However, a drawback of this method is that because the glycans are conjugated at separate spatially distinct locations, information on the synergistic or cooperative heteroglycan binding of different glycans cannot be determined. Further, due to spatial considerations, the glycans do not conjugate to these arrays at a density sufficient for cooperative binding of homoglycans, so the derived binding constants for the glycans can be distorted.

[0012] Boving and Hogersson teach the display of glycans on fluorescent microbead carriers and their analysis by multiplex flow cytometry suspension assays. Wang et al. extended this method to hundreds of glycans. The display of macrobeads is conceptually identical to the above bead-based library (006). Such libraries have limitations because the beads sterically interfere with the target and prevent the identification of synergistic binding and in some cases non-synergistic interactions.

[0013] Flitch et al. teach the display of glycan molecules on DNA molecules, but the method of using this monovalent library to encode the multivalent presentation of carbohydrates at a regulated density, which is often required for protein-carbohydrate interactions, is not clear.

[0014] There is a need to provide effective methods for identifying molecules for drug discovery, diagnostic development, and basic research studying protein-ligand interactions.

[0015] This background information is provided for the purpose of creating known information that the applicant believes may be relevant to the present invention. It is not necessarily intended to, nor should it be construed to, admit that any of the foregoing information constitutes prior art to the present invention. SUMMARY OF THE INVENTION

[0016] Generally speaking, the present invention may include a method for identifying a ligand or combination of ligands that binds to a target molecule, particularly a method for determining whether a ligand binds synergistically with a biomolecule of interest.

[0017] In one aspect, the present invention may include a method for generating a genetically encoded library of molecules displayed on nanoscale objects of the same composition ("silent carriers"), and the use of these libraries for elucidating protein-ligand interactions.

[0018] In one aspect, the present invention may include a method for identifying one or more molecular interactions between at least two ligands and a target molecule, the method may include the following: a) providing a plurality of silent carriers, each containing one of a plurality of unique nucleic acid codes, and each silent carrier being externally chemically identical; b) attaching a first ligand to a set of silent carriers containing a first nucleic acid code to form a first set of carriers; c) repeating step (b) to generate N sets where N ≧ 2, each set containing a different ligand or a different density of ligand, and each set containing a different nucleic acid code; d) pooling the N sets to form a first mixed library; e) contacting the first mixed library with the target molecule and identifying ligands that bind to the target molecule.

[0019] In some embodiments, the method further comprises pooling a set of binding ligands, excluding one set of one binding ligand to form a second mixed library, contacting the second mixed library with the target molecule; and determining, in the absence of the excluded ligand, which binding ligands have a lower or higher affinity for the target molecule.

[0020] Because the coding portion or silent carrier is of nanoscale size, there is less steric interference and it may be more suitable for the general discovery of synergistic binding. When the composition of the carrier is the same, the possibility of having undesirable molecular interactions with the target, which could complicate the analysis, is reduced.

[0021] In some embodiments, the silent carrier is a virus or phage. The plurality of nucleic acid codes can include degenerate DNA sequences of a portion of a viral or phage protein and / or unique fluorescent or enzyme detection markers.

[0022] In some embodiments, the ligand is a peptide, carbohydrate, or any other biomolecule. The target molecule can be a protein or other biomolecule, cell, organ, or any organic or inorganic material. In a preferred embodiment, the ligand comprises a glycan and the target molecule comprises a lectin.

[0023] In some embodiments, identification of the binding ligand is performed by extracting nucleic acids from a carrier containing the ligand bound to the target, and amplifying and sequencing the nucleic acids. Quantitative evaluation of ligand binding can be evaluated by the copy number after PCR. Alternatively, or additionally, identification of the binding ligand is performed by detecting a fluorescent or enzymatic detection marker, such as a reporter protein encoded in the DNA of the carrier, such that the detection marker is expressed by the host organism upon infection of the carrier. The reporter protein can include galactosidase, chloramphenicol acetyltransferase, or a fluorescent protein, or any other reporter protein or selectable marker known to those skilled in the art.

[0024] In some embodiments, identification of the binding ligand includes separating the target molecule-ligand-silent carrier complex in a pull-down assay, which can include steps of binding to a solid support, precipitation, centrifugation, magnetic capture, partitioning into another solvent, or any other separation method known to those skilled in the art.

[0025] In some embodiments, the first mixed library is a liquid mixed library, the target molecule is contained in a liquid, the target molecule is converted to a solid form, and is separated from the liquid mixture together with the ligand that binds to the target molecule. The target molecule can be a solution, dispersion, emulsion in a liquid, or the liquid itself. In one embodiment, the target molecule is a salt precipitated from a solution such as calcium carbonate. In one embodiment, the target molecule aggregates to insoluble particles. In one embodiment, the target molecule is converted from a liquid phase to a solid phase, such as water that changes to ice.

[0026] In another aspect, the present invention can include a method of displaying a ligand on a virus encoded silent by a nucleic acid code, the nucleic acid code being a degenerate sequence encoding a part of a native coat protein, or existing in a region of the viral genome that does not encode a protein, or existing in a region of the viral genome that encodes a viral held on top non-peptide. Thereby, the virus held on topThis display technology can be used with a virus that is incompatible with display technologies that require the expression of foreign proteins.

[0027] The carrier may be chemically modified to display a specific ligand on the surface of the carrier at a specific ligand density per carrier particle. Thus, the unique nucleic acid code within the carrier can identify either or both of a) the identity of the ligand and / or b) the density of the ligand displayed on the carrier. After mixing a first mixed library with a target molecule, the bound ligand can be separated from the unbound ligand, and subsequently the nucleic acid can be purified from the bound ligand carrier. The nucleic acid can then be amplified, for example, by use of PCR, and the evaluation of ligand binding can be performed by the copy number of the unique nucleic acid code.

[0028] In another aspect, the invention may include a method of calibrating a library of molecular targets, comprising adding a carrier (such as a phage) modified with a known ligand to a library (control phage), and subsequently screening the library with the ligand of the control phage. The known ligand may be a peptide, carbohydrate, or any biomolecule.

[0029] According to certain aspects, the present application provides a "silent carrier" that is a virus or bacteriophage virion of the same external chemical composition, preferably containing nucleic acid code including degenerate DNA tags within the genome packaged inside these particles. The genome of the virus or phage can be engineered in a way that does not result in a change in the chemical composition of the virion coat, such as through the use of degenerate codons in the coding regions of the virion coat, changes in DNA sequences encoding excised sequences, changes in DNA sequences that do not encode expressed protein sequences, or changes in DNA sequences encoding components not incorporated into the virion coat. Thus, a carrier library can be provided that includes multiple carriers (such as phages or viruses), all of which are externally chemically identical before binding to any ligand, but contain different nucleic acid molecules that are silently encoded therein.

[0030] Accordingly, the present invention can provide a library that facilitates the discovery of "synergistic" interactions where two or more molecules can bind simultaneously to one target. Such synergistic binding is generally known to enhance binding affinity compared to the interactions of individual ligands. According to another aspect, the present application provides a method for identifying protein-ligand interactions that can be used to more clearly understand whether the interaction between any given ligand and a protein from the library is synergistic or non-synergistic with respect to other ligands present in the same library.

[0031] The screening of synergistic binders described herein is optimal for "manually mixed libraries", such as those generated by silent coding or RNA / DNA tagging techniques and subsequent mixing. Such screening may be applicable to expression display libraries such as phage display libraries of peptides or proteins, or mRNA or DNA display libraries of polypeptides. Unlike "silently encoded" chemical library techniques, the creation of new libraries containing only defined components and / or lacking one of the components requires a great deal of effort. An example of the large-scale synthesis of specific combinations of DNA is known as array synthesis and library re-expression. These involve procedures that are more cumbersome than simply mixing N or a small number of M components from a pre-tagged set.

[0032] For a better understanding of the present invention, as well as another aspect and its further features, reference is made to the following description used in conjunction with the accompanying drawings:

Brief Description of the Drawings

[0033]

Figure 1

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[0038]

Figure 6

[0039] Figure 6H. Representative example of the pull-down of a glycan array of 74 glycans by the plant lectin UGA, which recognizes Fucα1-2-modifications and anti-Gal4 antibodies.

[0040]

Figure 7

Mode for Carrying Out the Invention

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0042] As used herein, "silent carrier" can include substantially all available viral genera of viruses, including those that cannot be engineered to produce a display or that cannot be effectively engineered by currently available recombinant DNA techniques, which viruses include "silent coding" of nucleic acid codes that are not expressed or on the surface of the virus where peptides are not displayed. Thus, the silent carriers of the present invention do not require the presence of a cloned peptide library or the introduction of new DNA segments or peptide variable regions. Suitable viruses include phages, but other viruses may also be included.

[0043] Conventionally, a "silent barcode" is placed in proximity to a variable region that is a foreign DNA fragment, enabling simultaneous characterization of these two regions by DNA sequencing. However, a "silent barcode" can be introduced anywhere within the phage genome, including the translation active region and the silent region, auxiliary proteins not used in phage assembly, or sequences excised from phage proteins (such as leader peptides).

[0044] In the present invention, viruses and bacteriophages also do not need to be derived from genera known to be amenable to efficient DNA manipulation. A host organism that produces such a virus only needs to take up modified DNA to an extent sufficient for the production of at least one modified particle. An example is the modification of the DNA of an archaeal virus by synthesis and heterologous manipulation in an E. coli host to introduce redundant codons into the coding region. When this DNA is reintroduced into an archaeal host, the efficiency is very low, but a set of silent archaeal viruses with the same coat composition but different DNA compositions within the genome can be produced. Once produced, such silent viruses can potentially propagate through reinfection of their host and do not require further recombinant DNA technology for scale-up production.

[0045] As described herein, suitable silent carrier embodiments typically do not use variable regions or contain foreign DNA fragments, and thus do not prioritize the position of the nucleic acid code used for "silent coding". As a result, the nature of the phage or virus used as the silent carrier is irrelevant. Thus, in certain embodiments, the carrier does not require a virus compatible with display technology, such as a virus that is susceptible to manipulation via recombinant DNA technology. For example, plant virus classes are known to be unable to tolerate the display of foreign sequences because their host plant cells proteolytically cleave almost all foreign peptide sequences during virus particle production. These sequences that are not cleaved can significantly interfere with particle packaging. These viruses can be used as silent carriers according to this application because they can introduce silent DNA codes into the native protein sequences of DNA regions that do not produce sequences. Since these changes do not result in changes in the external chemical composition, they are not excised and usually do not interfere with assembly. The virus is not modified from the outside.

[0046] In certain embodiments, silent coding can involve the production of a silent carrier that utilizes DNA codes in regions of the genome that encode entities that are not present in the assembled particles, such as the coding DNA of the coat protein itself, regions of DNA that do not encode proteins, or spliced RNA sequences or peptide leader sequences that are excised after translation.

[0047] This silent coding enables the expression in the laboratory of not only M13 phage or other phages that enable display, but also most known plant, animal, and archaeal viruses, as well as the operation in bacteriophages that do not allow the display of foreign peptide sequences on the coat protein. Silent variations can still be incorporated into the DNA or RNA of these bacteriophages or viruses to produce particles with the same external chemical composition but with different nucleic acid codes inside.

[0048] Carriers encoded silently can be chemically modified with ligands, such as by using standard protein ligation strategies known in the art. Multiple such carriers modified with different ligands and encoded with different nucleic acid codes can be mixed together to create a silently encoded mixed library of molecules of a desired composition. The methods presented herein describe the creation and utility of such libraries for discovering synergistic and non-synergistic interactions between these ligands and a target of interest through simple screening.

[0049] In some embodiments, the ligand can attach to the carrier by forming a covalent amide bond with a lysine or the amino terminus of the carrier coat protein. The carrier coat protein is modified to introduce a reactive handle that reacts with a homoreactive handle on the ligand. The homoreactive handle should not react with other functional groups of the coat protein. For example, the reactive handle is a strained alkyne and the homoreactive moiety is an azide.

[0050] In certain embodiments, the methods described herein can also function with mixtures of molecules tagged with DNA or RNA. In such screening, the nucleic acid "information tag" is not hidden within the bacteriophage capsid. As a result, this is not "silent" and may be less desirable because it can interact with ligands or target molecules.

[0051] In certain embodiments, a collection of different bacteriophage particles is provided, each carrying a unique nucleic acid code that functions as a "silent barcode." They are produced separately and modified with different molecules, such as glycan modifications. Pooling these modified libraries together creates a mixed library. In a preferred embodiment, the mixed library includes a "liquid glycan array" that can track glycan modifications by sequencing the silent barcodes.

[0052] In certain embodiments, the method provides a display on a "silent carrier" that involves mixing N different sets of silent carriers, where each set has a different ligand or a different density of ligands. In certain embodiments, the different sets of ligands are mixed in equal ratios to simplify downstream analysis, although other relevant ratios may be implemented. Using this mixture that contains N different ligands in the same solution, one-step selection is performed with a target molecule to identify a subset of M potential ligands. Standard selection methods known in the art, such as pull-down assays and next-generation sequencing of the isolated mixed DNA molecules, can be used to identify the nucleic acid codes associated with the ligands that bind to the target. The M identified molecules have some affinity for the target molecule and can be "synergistic binders" or "non-synergistic binders." For example, a collection of m molecules is a set that includes molecules M1, M2, M3,....Mm (i.e., when m = 10, M1, M2, M3, M4,....M10). The set of m molecules can be designated as {M}. Thus, there can be a set {M} and subsets {M-Mi}, where the subset {M-Mi} is the set {M} excluding one set member Mi.

[0053] In this particular embodiment, when a liquid glycan array having N different glycans is mixed with, for example, a protein of unknown carbohydrate-binding properties and then the protein is pulled down, M bound glycans (M1, M2, M3, etc.) are enriched. To test whether M1 is a glycan that acts synergistically with components M2, M3, etc., a mixed subset of the same set excluding all M glycans and glycan M1 ( "M-M1") is created. Pull-down of these mixtures identifies whether glycan M1 acts synergistically or antagonistically with other glycans. Since the process is simple mixing, this mixing and pull-down can be repeated m times to clearly identify all interactions as either "synergistic" or "non-synergistic".

[0054] "Pull-down" assays include those in which one or the other of a ligand and its target is immobilized or bound to a solid support such as beads, facilitating the separation of the bound carrier-ligand-target complex from unbound ligand. For example, a hexahistidine tag can be provided on the target molecule and on a hexahistidine-binding molecule such as nitrilotriacetic acid (NTA) on the beads. Other possibilities include biotinylation of a protein and streptavidin beads; or Fc fusion of a protein and protein G beads. It is known to form bonds under heterogeneous reaction conditions, and two reactants known as "bioorthogonal ligation" can be used; an example is the pair of tetrazine and trans-cyclooctene of a bioorthogonal reaction component: tetrazine can be placed on the protein and cyclooctane can be immobilized on the beads. Another example is the use of cyclooctene and azide: cyclooctene can be used to functionalize the protein and azide can be placed on the surface of the beads. However, these are part of a non-exhaustive list of examples and are intended to illustrate other processes that are also suitable for use in pull-downs with specific, strong and complementary covalent or non-covalent interactions.

[0055] Following pull-down screening, identification of the nucleic acid code involves deep sequencing or next-generation sequencing. For example, if beads are used in the selection step, the beads are exposed to biochemical extraction conditions, the DNA material is separated from the beads, and the extracted DNA is subjected to polymerase chain reaction that amplifies the extracted DNA and attaches a novel sequence called an "adapter" sequence to the extracted DNA, thereby enabling sequencing of this DNA using next-generation sequencing technologies such as Illumina or Ion Torrent. Post-processing, PCR, or incorporation of the adapter sequence is an optional step; one example is the conversion of phage genomes into DNA corresponding to Illumina sequencing. Another example includes modification of Illumina sequencing technology that uses existing phage DNA as an adapter. Alternatively, Illumina adapters may be present in phage DNA. Using both examples of changes to the procedure, the need for a PCR step or other steps introducing an "adapter sequence" can be reduced. Separation of DNA from the beads may vary if different genera of bacteriophages are used or different downstream DNA processing methods are used (e.g., PCR using specific reagents). Such separation of DNA from the beads can be readily optimized according to methods known in the art. Following PCR, if the copy number of DNA molecules associated with a particular nucleic acid code exceeds a minimum threshold or ratio, a suitable "hit" is identified.

[0056] In certain exemplary embodiments, the present application provides for the use of a "liquid"-based format for glycan arrays. In a liquid-based format, multiple glycans attached to freely diffusing silent carriers, such as phages, simultaneously bind to target biomolecules, enabling both hetero- and homoglycan binding to occur cooperatively. For the liquid format to function, typically, for example, a method is needed to determine which glycan is bound to the target. Thus, the present application provides, in certain embodiments, for the construction of arrays using silent encoding of glycans; thereafter, a collection of chemically identical particles is modified with different glycans and then mixed together to form a mixture of N glycans in the same solution. Using this mixture containing N glycans in the same solution, a one-step selection consisting of pull-down and next-generation sequencing of isolated mixed DNA molecules is performed to identify an enriched subset of M potential binding glycans. Standard selection methods known in the art may be used. In this example, the M glycans identified are presumptively referred to as "synergistic binders" or "non-synergistic binders."

[0057] Next, in a single step, determine whether any one of the ligands identified from the subset of {M} is a synergistic ligand or a non-synergistic ligand. For example, to determine whether a ligand Mi from the set of {M} ligands is a "synergistic binder" or a "non-synergistic" binder, construct a novel mixture containing {M} and {M-Mi} components (the latter lacking the ligand Mi). Repeat the enrichment process respectively to identify the ligands enriched in the presence or absence of component Mi. Then, compare the copy numbers of each ligand drawn from the {M} and {M-Mi} sets. After pulling down from each set, if the ligands show the same copy number, they are defined as "non-synergistic". Conversely, if the copy numbers of the ligands in the two mixtures are significantly different, the ligand is defined as "synergistic" (or perhaps antagonistic) with component Mi. Molecules for which the enriched fractions are not statistically significant between the two experiments do not act synergistically with molecule Mi and can be used to identify synergistic binding interactions by showing a lack or severe decrease in binding in the absence of molecule Mi.

[0058] Thus, a library of N ligands is reduced to M subsets, and a series of selection steps in which each member of {M} is sequentially excluded provides the synergistic binding ability of each member of {M} with each other member of {M}. Approximately N 2 Unlike screening with a separated molecular library of a molecular array scaled as approximately N, this screening requires only M+1 screenings where M is a number significantly smaller than N.

[0059] In certain embodiments, the present invention includes a method for determining the glycan priority of a lectin of interest as a drug discovery target. Other classes of molecules can be similarly examined as target molecules. Techniques similar to those of "glycan arrays", such as protein arrays, peptide arrays, small molecule arrays, nucleic acids, and similar arrays, are known. They are produced and used in the same manner as glycan arrays and can be used in the methods herein with minor or no conceptual changes.

[0060] In an exemplary embodiment, the glycan chemically binds to the filamentous phage M13, such as via the N-terminus of g8p or via an exposed lysine residue located at position 8 of g8p. Similar chemical modifications to plant viruses, animal viruses, or archaeal viruses can also be used. This application provides for the production of N separate preparations, each encoding N glycan variants of those viruses that are silent encoded by the nucleic acid code. The variants are mixed to form a mixed library, which may be a liquid array of N glycans. The remaining selection process for identifying an enriched subset of potentially M ligands, which is a pull-down of the modified virions and next-generation sequencing of the isolated mixed DNA molecules, is described herein.

[0061] In certain embodiments, an example of the method of the present invention can provide for the use of a target having a known binding affinity and known synergistic interactions to calibrate the system. For example, a liquid array of N glycans is combined with a known calibration target and the same array is mixed with an unknown target. Using the same "pull-down" assay for each, separation beads having glycans that bind to the calibration target and the unknown target are obtained. By comparing the copy numbers of hits from each of the control target and the unknown target, information regarding the relative binding affinity of the unknown target can be provided.

[0062] In certain embodiments, examples of methods can be used to measure multivalent and homoglycan binding by encoding carriers with glycans at different densities. In one example, the M13 phage carrier contains approximately 2700 copies of g8p per particle, which means that 1 to 2700 glycans per particle can be labeled. By varying the ratio of the chemical crosslinking agent to the particles in the crosslinking reaction, the average amount of the crosslinking agent per particle can be adjusted. Thus, in subsequent glycan binding reactions, if the amount of glycan provided is greater than the available crosslinking agent, the average number of glycan moieties crosslinked to the particles results in different display densities. By creating several different libraries that display the same glycan but at different densities with different nucleic acid codes, the effect of multivalent and homoglycan cooperative binding can be measured.

[0063] To obtain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Accordingly, they are in no way intended to limit the scope of the invention.

[0064] Examples Example 1: Cloning and Isolation of Silent SDB and SVEK Libraries

[0065] The silent-encoded phage library was cloned using the following procedure. The silent distal barcode (SDB) region (Figs. 1 and 6A) was introduced into M13KE using PCR amplification followed by NEBuilder HiFi DNA Assembly (NEB). The insert fragment was PCR amplified using primers 1 5’-GAG ATT TTC AAC GTG AAA AAA CTN CTN TTY GCN ATH CCN CTN GTG GTA CCT TTC TAT TCT CA-3’ and primer 2 5’-TTA AGA CTC CTT ATT ACG CAG TA-3’, and the vector fragment was PCR amplified using primer 3 5’-TTG CTA ACA TAC TGC GTA ATA AG-3’ as the forward primer and primer 4 5’-TTT TTT CAC GTT GAA AAT CTC-3’. Phage-derived dsDNA based on the M13KE clone containing the stuffer sequence CAG TTT ACG TAG CTG CAT CAG GGT GGA GGT corresponding to the peptide QFT*LHQGGG was used as a template, where * represents a stop codon. The PCR amplified fragment was treated with the restriction enzyme Dnp1 and then gel purified. Next, NEBuilder Hifi DNA Assembly was performed according to the manufacturer's instructions. The resulting ligated DNA was transformed into Escherichia coli K12 ER2738 and grown overnight at 37°C. The overnight culture was then centrifuged to separate the bacteriophage from the host cells. The host cells were then treated with a MiniPrep kit to extract the dsDNA for subsequent cloning rounds. To clone the silent-encoded SVEKNDQKTYHAGGG, the following primers were used to introduce the peptide. The insert fragment was PCR amplified using primer 5 5’GTG GTA CCT TTC TAT TCT CAC TCG AGY GTN GAR AAR AAY GAY CAR AAR ACN TAY CAY GCN GGN GGN GGN TCG GCC GAA ACT GTT GAA AG-3’ as the forward primer and primer 2.The vector fragment was PCR amplified using primers 4 and primer 6 5’-CGA GTG AGA ATA GAA AGG TAC-3’. Except that the resulting ligated DNA was transformed into E. coli SS320 cells (Lucigen), the PCR fragments were processed in the same manner as before using NEBuilder Hifi DNA assembly. The resulting overnight culture was centrifuged to remove host cells and PEG precipitated to concentrate the released phage. The PEG precipitated phage was resuspended in 1×PBS 50% glycerol and stored at -20 °C. For silent coding in the SDB region, 6.0×10. 3 results in possible combinations of sequences, while in the SVEK region, 4.2×10 6 results in possible combinations of sequences. Combining the maximum spaces of the SDB - SVEK library results in 2.6×10 10 possible combinations of sequences. Monoclonal and silently - encoded phages were isolated by plaque purification. Phages were seeded at a density of 100 plaques per plate and individually picked. Phage isolates were selected, grown, and sequenced.

[0066] To increase the accuracy of next - generation sequencing, the Hamming distance was defined as the number of changes required to convert a string from one sequence to another, and only barcodes with a Hamming distance greater than 3 were retained. Unique barcoded phages were amplified and concentrated using PEG precipitation.

[0067] Figure 1 provides a scheme related to the construction of silent barcodes within the g3p leader peptide sequence.

[0068] Table 1 provides examples of DNA sequences of phage isolates containing silent distal barcodes (SDBs) described in FIG. 1. The SDB is a shortened string that contains only degenerate changes in DNA codons, but the codon sequence of the SDB region contains the complete DNA sequence of the SDB region. When the codon sequence of the SDB region is compared with the SDB sequence, it is determined that it corresponds to the SDB for every three DNA bases of the codon sequence.

Table 1-1

Table 1-2

[0069] Example 2: Construction of Fluorescent Phage Controls for Colorimetric Evaluation of Panning Protocols

[0070] The fluorescent phage is a derivative of the filamentous phage vector M13Ke and has the fluorescent proteins mCherry and mNeonGreen cloned in place of the lacZα fragment (Figure 6A). These phages produce plaques that fluoresce when irradiated with light of the correct wavelength. Phages expressing the fluorescent proteins were constructed using the following procedure. The insert fragment was PCR amplified using primer 7 5’-GCG GAT AAC AAT TTC ACA CAG GAA ACA GCT ATG GTG AGC AAG GGC GAG-3’ and primer 8 5’-TTA AAT TTT TGT TAA ATC AGC TCA TTT TTT ACT TGT ACA GCT CGT CCA-3’. The vector mCherry-pBAD was used as the template for the mCherry insert, and the vector mNeonGreen-pBAD was used as the template for the mNeonGreen insert. The vector fragment was PCR amplified using primer 9 5’- AAA ATG AGC TGA TTT AAC AAA AAT TTA A-3’ and primer 10 5’- AGC TGT TTC CTG TGT GAA AT-3’. An M13KE derivative containing the SDB sequence CTT CTA TTT GCT ATT CCT CTA was used as the template for the vector PCR of the mCherry construct, and a derivative containing the SDB sequence CTA CTG TTC GCA ATC CCG CTA was used as the template for the mNeonGreen construct. Both templates are M13KE derivatives containing the stuffer sequence CAG TTT ACG TAG CTG CAT CAG GGT GGA GGT corresponding to the peptide QFT*LHQGGG in the peptide region. To ensure accuracy, isolated phage plaques were selected, amplified, and sequenced.

[0071] Next, the fluorescent phage was further modified to express the peptide SWYDLYHGGG. To do this, an insert fragment was generated using primer 9 5’-TA GTG GTA CCT TTC TAT TCT CAC TCG AGY TGG TAY GAY CTN TAY CAY GGN GGN GGN TCG GCC GAA ACT GTT GAA-3’ and primer 2. The vector fragment was generated using primers 4 and 6 using M13 mNeonGreen and mCherry as templates. After purification, the fragments were ligated using NEBuiilder HiFi and cloned into E. coli 10G F’. Primer 9 contains a degenerate sequence 2×(AGY)1×(TGG)2×(TAY)2×(GAY)4×(CTN)2×(TAY)2×(CAY)4×(GGN)4×(GGN)4×(GGN) that results in a library of 8192 possible sequences. Since the SDB of the fluorescent phage is fixed, this allows for the individual identification of specific phages by sequencing. The resulting phages were isolated and sequenced.

[0072] To increase the accuracy of next-generation sequencing, the Hamming distance was defined as the number of changes required to convert a string from one sequence to another, and only barcodes with a Hamming distance greater than 3 were retained. Unique barcoded phages were amplified and concentrated using PEG precipitation.

[0073] Example 3: Cloning of M13 blocking phage

[0074] The blocking phage is an M13 derivative and contains silent mutations within the primer region of Illumina. This means that the Illumina sequence is invisible because the primers used to amplify the peptide region of the phage do not bind to the blocking phage genomic DNA and cannot be PCR amplified. The M13 blocking phage was constructed using the following method. M13 dsDNA was used as the template for both PCR reactions. The vector was amplified with primer 10 5’- CAG AAA ATT CAT TTA CTA ACG TCT GGA A - 3’ and primer 11 5’- AAA GGA ACA ACT AAA GGA ATT GCG- 3’. The insert was amplified using forward primer 12 5’-TAT TCG CAA TTC CTT TAG TTG TTC CTT TGT ACA GCC ATA GTG CGG AGA CCG TGG AAA GTT GTT TAG CAA AAC CCC A-3’ and primer 13 5’-TAA ATG AAT TTT CTG TA-3’. The insert fragment and vector fragment were treated with Dpn1 and the purified fragments were subjected to NEBuilder Hifi assembly and gel purified before transformation into E. coli XL1 Blue. The isolated plaques were sequenced to ensure accuracy.

[0075] Example 4: Binding of glycans to filamentous phage.

[0076] Binding of glycans to filamentous phage virions was achieved using a two-step procedure and a propargyl-N-hydroxysuccinimide or dibenzocyclooctyne N-hydroxysuccinimide (DBCO-HNS) linker (Figures 2 and 7A and 7B).

[0077] Figure 7A generally shows a chemical ligation strategy scheme used to incorporate azidoglycan into the phage pVIII protein and create a glycan liquid array. Each reaction can produce phages containing modified and unmodified p8 proteins. A two-step reaction can produce a fully modified product ("prod"), a partially modified intermediate ("int."), or an unreacted p8 protein ("s.m"). The modification density of the phage is determined by the ratio of these species. Figure 7B shows a chemical schematic of the amino acid sequence of the p8 protein, modification with dibenzocyclooctyne N-hydroxysuccinimide (DBCO-HNS) linker, and ligation of the azido linker and glycan abnormally bound to the DBCO-modified p8 protein.

[0078] First, phages with a single silent barcode are reacted with a linker via an N-hydroxysuccinimide group. This linker covalently attaches to the phage virion by the major virion coat protein pVIII via either the N-terminus of the pVIII polypeptide sequence. Then, click chemistry between a propargyl group and an azide derivative of the carbohydrate that covalently attaches the carbohydrate to the phage virion is used to react the phage with a crosslinker. Glycan β-azidomannoside was used to optimize this chemical reaction. The phages were first incubated for 30 minutes with 1x, 20x, and 50x equivalents of dibenzocyclooctyne-sulfo-N-hydroxy-succinimidyl ester (the equivalents were calculated with respect to the molar concentration of the total pVIII protein per phage). For example: 10 12 phages contain 2700*10 12 pVIII proteins per mL, which is

Number

[0079] The phage pVIII protein contains two solvent-exposed amino groups that can be modified (Figure 7B). The first is the N-terminal amino group, and the second is lysine (the 8th from the N-terminal). MALDI analysis with a sinapic acid matrix (the matrix also contains TFA) showed an additional peak at approximately 4850 (m / z), which remained the same after modification. The results are shown in Figure 7E. Previous studies by several groups have shown the acid sensitivity of the peptide bond between aspartic acid (D) and proline (P); however, the phage pVIII protein contains a D-P bond. Cleavage of this bond produces two fragments with masses corresponding to 505 and 4833. Since the mass of the latter peak did not change after glycan modification, it was concluded that all modifications were carried out at the N-terminal amino group.

[0080] Example 5: Encoding of Different Multivalent Densities of Ligands Using a Silent Carrier

[0081] The density of the display on the phage particles can also be encoded. Figure 3 provides an illustration of the effect of modulating the crosslinking chemistry to produce particles displaying the same glycan at different densities.

[0082] As depicted in Figure 3, when each silent carrier phage is conjugated with a different glycan or a different density of glycan, a mixture of different silent carriers is produced. When the mixtures are pooled together, a mixture is produced where each glycan and each different glycan density is associated with a unique carrier that can be identified by the silent barcode. As shown in Figure 3, P1 is bound to glycan 1 at a density of 3 glycan molecules per phage particle. P2 is bound to glycan 2 at a density of 3 glycan molecules per phage particle. P3 is bound to glycan 2 but at a density of 5 glycan molecules per phage particle, and P4 is bound to glycan 2 at a density of 10 glycan molecules per phage particle.

[0083] Example 6: Demonstration of Glycan Modification Using ELISA

[0084] To demonstrate that the modification of the phage does not destroy the glycan, an ELISA-based conformation of glycan binding was performed. ELISA was completed according to the published protocol. First, the microtiter plate was coated overnight with a dilution gradient of gal4-phage, as well as unmodified phage and linker phage for negative control in PBS. The plate was then washed and incubated for 2 hours with 100 μL of a solution containing 1 μg / ml anti-Gal4 antibody. The plate was then washed again and incubated for 40 minutes with the secondary antibody, goat anti-mouse HRP-tagged (1:5000 dilution). The plate was then washed and HRP substrate TMB was added. After construction, the reaction was stopped with 1(M) phosphoric acid, read at 450 nm, and the data collected was processed with Origin software.

[0085] Figure 4 shows the specific recognition of glycan phages adsorbed on a polystyrene plate by a glycan-specific monoclonal antibody. Unmodified phages and DBCO phages are used as controls. Each data point represents the average value of three replicates.

[0086] Figure 5 shows the recovery rate of glycans from the library detected by deep sequencing. The three glycans used in the mixture are tetragalactofuranose (gal4), beta-mannose (man), and lactose (lac). As targets, mouse mAbs produced against the corresponding known targets - gal4, ConA, and galectin 3, respectively, were used. The enrichment of each barcode is calculated by dividing the number of reads detected in the elution sample by the corresponding number in the input sample. The dotted line in each panel indicates an enrichment of 1-fold.

[0087] Example 7: Panning of Glycan-Binding Proteins in Solution

[0088] Untagged glycan-binding protein (GBP) was first chemically modified with NHS-PEG4 biotin, while the glycan-binding antibody was not modified. Next, a liquid glycan array (LiGA) was mixed with 10 μg of glycan-binding protein at room temperature for 1 hour. To capture GBP and bound LiGA phage, 10 μL of either protein G beads pre-washed with binding buffer for biotinylated GBP or glycan-binding antibody was added. Additionally, at this point, 0.1% BSA was added to the mixture as a blocking agent to reduce non-specific binding of phage to the beads. After a 45-minute incubation, the mixture was centrifuged at 500 g for 1 minute to collect the beads. The supernatant was discarded, and the beads were washed with 1 mL of PBST buffer and centrifuged to collect the beads. The washing step was repeated three times. If the experiment was conducted as an optimization experiment to evaluate glycan binding, acid elution was used to elute the phage from the beads. To do this, the beads were mixed with 0.2 M glycine buffer pH 2 for 10 minutes; then, the solution was neutralized with 1 M Tris pH 9, and the eluted phage was counted by phage plating. Since LiGA (Figure 6C) contains fluorescent phage (mNeonGreen) conjugated to mannose and fluorescent phage (mCherry) conjugated to galactose, these controls were used to demonstrate binding to mannose- and galactose-binding lectins. This allowed the optimization of the panning procedure without performing deep sequencing (Figure 6C). Optimization of panning with concanavalin A (ConA) showed that mNeonGreen phage displaying mannose was retained in higher amounts than mCherry phage displaying galactose. This result is consistent with ConA being a mannose-binding lectin.

[0089] For samples subjected to deep sequencing, the beads were resuspended in 30 μL of Tris-EDTA buffer (Tris 10 mM + EDTA 0.01 mM pH). Next, 30 μL of hexane was added to the beads, and the mixture was shaken and incubated at room temperature for 10 minutes to decompose and release the phage genomic DNA. Then, the hexane was evaporated by incubating at 68 °C for 8 minutes. After evaporation of the solvent, the beads were pelleted by centrifugation at 21,000 g for 2 minutes. The remaining supernatant was subjected to PCR amplification to amplify the SDB-SVEK region and attach the Illumina deep sequencing adapter. Deep sequencing with Ulex Europaeus Agglutinin (UEA lectin) showed that phages displaying glycans with terminal or branched fucose were retained (Figure 6E), and panning with anti-Gal4 antibody showed retention of Gal4-displaying phages.

[0090] Figure 6A shows a schematic diagram of the genome of bacteriophage m13 and the locations for introducing silent barcodes. The barcodes can be in the translated regions of coat proteins such as p3. They can also be inserted into regions that do not encode phage proteins (lower reporter box). Such genes do not exist as protein products in the phage but are transduced into the host organism by the phage. Figure 6B shows that different reporter proteins can be used to track different chemical modifications or the density of these same modifications. In the example, high density (1500 mannose molecules per phage), medium density (500 mannose molecules per phage), low density (200 mannose molecules per phage), and absence of mannose molecules are encoded by four different reporter proteins, respectively. Phages containing high mannose modifications transduce the mNeonGreen gene into host bacteria and form green fluorescent plaques. Similarly, on agar containing the colorimetric substrate X-gal, medium Man phages transduce the mCherry protein and form red plaques, and low density Man-phages transduce the α-galactosidase (α-Gal) gene and form blue plaques. Phages that do not display glycans do not transduce the reported ones and form white plaques. The ratio of green, red, blue, and white plaques before and after selection can be used to monitor the effect of mannose density on enrichment for a specific target.

[0091] Figure 6C shows that a colorimetric or fluorescent reporter can be combined with a silent barcode that is analyzed by sequencing. In this example, two mixtures are generated: LiGA1 contains nine different α-Gal(+) carriers modified with galactose, lactose, or LNT tetrasaccharide at three different densities (1500, 500, or 200 copies per phage). The nine combinations can be distinguished by sequencing of the barcode. LiGA1 also contains mannose 1500-mNeonGreen and lactose 1500-mCherry and an unmodified "blocking phage" that does not express the reporter. The extended LiGA2 mixture contains, in addition to everything included in LiGA1, an additional nine clones containing β-mannose, α-mannose, and α-Man3 glycan at three different densities.

[0092] Figure 6D shows that a four-color scheme monitored the enrichment of the LiGA1 mixture shown in Figure 6C on a polystyrene plate coated with the mannose-binding lectin ConA. The number of particles in the input and output was estimated by plaque-forming assay. Figure 6E shows that the recovery rate of particles containing high-density mannose detected as green plaques is 15%. The recovery rate of red particles containing lactose glycan that does not bind to ConA is 0.3%. Only 0.04% of the unmodified "blocking" phage particles were recovered; since this population does not show a ligand that binds to ConA, a similarly low recovery rate (0.06%) was observed for the nine phage clones of the α-Gal(+) "blue" population. Figure 6F shows the results of a similar experiment repeated with LiGA2, where the α-Gal(+) population contains a ConA-binding ligand. The recovery rate of the "blue" population is significantly higher than that observed in Figure 6E and ten times higher than that of the unmodified white phage. The recovery rates of the ConA-binding green clone and the non-ConA-binding "red" clone are similar to those observed in Figure 6D.

[0093] Figure 6G shows that using the same four-color scheme, the recovery rate of the library on any target, such as cells containing the mannose-binding lectin DC-SIGN, can be monitored and optimized. The initial population contains green and white plaques at a ratio of 1:100. After four washes, cell pellet p4 contains green (Man) and white (unglycosylated phage) at a ratio of 1:1, indicating that the Man phage was concentrated 100-fold. In cells without DC-SIGN, fewer Man-green phage clones are recovered. Sequencing of the DNA associated with the cell pellet confirms the enrichment, but colorimetric monitoring can be used to optimize the selection procedure without the need for sequencing.

[0094] Figure 6H shows a representative example of the pull-down of an array of 74 glycans by the plant lectin UGA, which recognizes Fucα1-2 modifications and anti-Gal4 antibodies. The specific glycans present in the array due to the Fucα1-2 modification are as follows. Te212: Fucα1-2Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4[Fucα1-3]GlcNAcβ-phage; Te222: GalNAcα1-3[Fucα1-2]Galβ1-4GlcNAcβ-phage (no enrichment detected: false negative?); Te223: Galα1-3[Fucα1-2]Galβ1-4GlcNAcβ-phage; Te224: GalNAcα1-3[Fucα1-2]Galβ1-4Glcβ-phage; Te118: Fucα1-2Galβ1-4[Fucα1-3]GlcNAcβ-phage (no enrichment detected); Te303: Neu5Acα2-3[Neu5Acα2-3Galβ1-3GalNAcβ1-4]Galβ1-4Glcβ-phage; Tr116: Fucα1-2Galβ1-3GlcNAcβ-phage

[0095] Example 8: Demonstration of Cell-Based Screening Using LiGA

[0096] Using LiGA, the glycan-binding properties of whole living cells can be evaluated. To demonstrate this, a LiGA array was panned against a rat 6 fibroblast stable cell line that highly expresses human dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin receptor (DC-SIGN). As a negative control, a Rat-6 fibroblast cell line that did not express any protein was used. The details of this cell line construction are described in [4]. DC SIGN is a C-type lectin that has an affinity for high-mannose and fucose-containing glycans. To perform this experiment, trypsin was used to detach the logarithmic-phase cells from the flask and resuspend them in Hepes buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 2 mM CaCl, 1% BSA) at 1×10 6 cells / mL. Then, 1 mL aliquots of the cells were pelleted (1000 rpm / 4 min) and resuspended in 500 μL of Hepes buffer containing 1×10 8 pfu of LiGA phage and 1×10 8 pfu of blocking phage. The LiGA array used in this example contains fluorescent phages for positive and negative controls to monitor the efficiency of washing (Figure 6G). The positive control phage was an mNeonGreen fluorescent phage conjugated to monovalent mannose. The negative control phage used was an mCherry fluorescent phage conjugated to galactose. The cells were then incubated on ice for 2 hours. The cells were then washed 3 times using 4 mL of Hepes buffer and resuspended in 30 μL of H2O. To analyze the phage titer, 5 μL of the sample was taken out before each washing step. After boiling the sample for 10 minutes, it was centrifuged at 21000g for 5 minutes to remove cell debris. The supernatant was then transferred to a PCR tube containing the PCR reaction mixture, and an amplification product for Illumina sequencing was prepared. Titration of the non-boiled samples showed that after washing the cells 3 times, the positive phage titer was 10-fold higher than that of the negative control phage (Figure 6G). Furthermore, the number of blocking phages was reduced from 1×10 8 pfu to 1×10 5It decreased to pfu. Even when the cells were further washed, the phage titer did not significantly decrease. Deep sequencing of the phages remaining bound to the cells showed that phages conjugated to mannose-containing glycans were retained in the panned population, while phages conjugated to other glycans were not retained.

[0097] Definitions and Interpretations The description of the present invention has been presented for purposes of illustration and description, but is not intended to be exhaustive or to limit the invention to the disclosed form. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Embodiments were selected and described in order to best explain the principles of the invention and its practical application, and to enable others skilled in the art to understand the invention for various embodiments with various modifications suitable for the intended particular use. To the extent that the following description relates to a particular embodiment or a particular use of the present invention, it is intended only as an illustration and is not intended to limit the invention claimed in the claims.

[0098] All means or steps and corresponding structures, materials, acts, and equivalents of the functional elements in the claims appended hereto are intended to include any structure, material, or act for performing the functions in combination with other claimed elements specifically claimed.

[0099] References to "one embodiment", "an embodiment", etc. in this specification indicate that the described embodiments can include a particular aspect, feature, structure, or characteristic, but not necessarily all embodiments include that aspect, feature, structure, or characteristic. Further, such phrases can, but not necessarily, refer to the same embodiment mentioned in other parts of this specification. Further, when a particular aspect, feature, structure, or characteristic is described in relation to an embodiment, combining, affecting, or connecting such an aspect, characteristic, structure, or characteristic with other embodiments is within the knowledge of those skilled in the art, whether or not such connection or combination is explicitly described. In other words, any element or function can be combined with other elements or functions of different embodiments unless there is an obvious or inherent non - compatibility between them, or unless specifically excluded.

[0100] It is further noted that the claims can be drafted to exclude any optional elements. Therefore, this description is intended to serve as a preamble for using exclusive terms such as "only", "solely", etc. in relation to the recitation of elements of the claims or the use of "negative" limitations. Terms such as "preferably", "preferably", "preferred", "optionally", "may", and similar terms are used to indicate that the referenced item, condition, or step is an optional (not essential) feature of the present invention.

[0101] The singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. The term "and / or" means any one of the items, any combination of the items, or all of the items to which this term is associated.

[0102] As will be understood by those skilled in the art, for any purpose, and particularly from the perspective of providing the described description, all ranges recited herein also include any and all possible sub-ranges and combinations of such sub-ranges, as well as the individual values that make up that range, particularly integer values. The recited ranges (e.g., weight percentages or carbon groups) include each specific value, integer, decimal, or identity within the range. It can be readily recognized that the recited ranges sufficiently describe and enable the same range to be divided into at least halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range described herein can be readily divided into sub-thirds, middle-thirds, and upper-thirds, etc.

[0103] As will also be understood by those skilled in the art, all ranges described herein, and all language such as "maximum", "at least", "greater than", "less than", "more", "above", etc., include the recited numbers, and such terms refer to ranges that can be divided into sub-ranges as described above. References JPEG0007714325000004.jpg132168 JPEG0007714325000005.jpg228168 JPEG0007714325000006.jpg44168

Sequence Listing Free-Text

[0104] Sequence Listing 1 <223>n is.a.c.g or t. Sequence Listing 8 <223>n is.a.c.g or t. Sequence Listing 43 <223>n is.a.c.g or t.

Claims

1. A method for identifying one or more molecular interactions between at least two ligands and a target molecule, comprising: a) providing a plurality of silent carriers, each containing one of a plurality of unique silent nucleic acid sequence codes, and all of the plurality of silent carriers having the same external chemical composition before any ligand binds thereto; b) attaching a first ligand to a set of silent carriers containing a first silent nucleic acid sequence code to form a first set of said silent carriers and said ligand; c) repeating step (b) to generate N sets where N ≧ 2, each set containing a ligand different from the ligands of other sets or a ligand having a density different from the density of the ligands of other sets, and each set containing a different silent nucleic acid sequence code; d) pooling the N sets to form a first mixed library; e) contacting said first mixed library with a target molecule to identify a plurality of ligands that bind to the target molecule; f) pooling a plurality of sets of silent carriers different from the plurality of ligands identified as binding to the target molecule, excluding one ligand identified as binding to the target molecule and one set of silent carriers, to form a second mixed library; g) contacting the second mixed library with the target molecule; h) determining, in the absence of the ligands excluded from the second mixed library, which plurality of bound ligands or which density of bound ligands has a lower or higher affinity for the target molecule; wherein the ligand comprises a glycan and the target molecule comprises a biomolecule or a cell expressing the biomolecule.

2. The method according to claim 1, wherein the carrier is a virus or a phage.

3. The method according to claim 1 or 2, wherein the plurality of silent nucleic acid sequence codes comprise degenerate DNA sequences of a part of a viral or phage protein.

4. The method according to any one of claims 1 to 3, wherein at least one silent nucleic acid sequence code encodes a distinguishable fluorescent or enzyme detection marker.

5. The method according to any one of claims 1 to 4, wherein the set of silent carriers comprises carriers chemically modified to display ligands on the surface of the carriers at a specific density.

6. The method according to any one of claims 1 to 5, wherein the identification of the bound ligand is performed by extracting nucleic acids from carriers containing ligands bound to the target, and amplifying and sequencing the nucleic acids.

7. The method according to claim 6, wherein the quantitative evaluation of the binding of the ligand is evaluated by the copy number after PCR.

8. The method according to claim 4, wherein the identification of the bound ligand is performed by detecting a fluorescent or enzyme detection marker.

9. The method according to claim 1 or 2, wherein the target molecule comprises a biomolecule.

10. The method according to claim 1, wherein the identification of the binding ligand comprises separating the target molecule-ligand-silent carrier complex in a pull-down assay.

11. The method according to claim 10, wherein the pull-down assay comprises steps of binding to a solid support, precipitation, centrifugation, magnetic capture, or partitioning into another solvent.

12. The method according to claim 4, wherein the detection marker comprises a reporter protein encoded in the DNA of the carrier such that the detection marker is expressed by the host organism upon infection of the carrier.

13. The method according to claim 12, wherein the reporter protein comprises galactosidase, chloramphenicol acetyltransferase, or a fluorescent protein.

14. The method according to claim 1 or 2, wherein the ligand is attached to the carrier by forming a covalent amide bond with lysine or the amino terminus of the carrier coat protein.

15. The method according to claim 14, wherein the carrier coat protein is modified to introduce a first reactive handle that reacts with a second reactive handle on the ligand, and the second reactive handle does not react with other functional groups on the coat protein.

16. The method according to claim 15, wherein the first reactive handle is a strained alkyne and the second reactive handle is an azide.

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