US-12618062-B2 - Display of molecules on silently genetically encoded nanoscale carriers for determining synergistic molecular interactions

Abstract

The present application provides a method of producing a “liquid” array of ligand (such as glycan) modified bacteriophage where the ligand modification is encoded genetically within the bacteriophage genome. This method will allow for the determination of the ligand binding profile of biomacromolecules and cells. Furthermore the method allows the elucidation of ligand-protein interactions where ligand binding is co-operative and synergistic.

Inventors

• Ratmir Derda
• Nicholas Bennett
• SUSMITA SARKAR

Assignees

• THE GOVERNORS OF THE UNIVERSITY OF ALBERTA

Dates

Publication Date

20260505

Application Date

20180131

Claims (20)

1. 1 . A method of identifying one or more molecular interactions between at least two ligands and a target molecule, the method comprising: (a) providing a plurality of silent carriers, each comprising one of a plurality of unique nucleic acid codes therein, wherein each silent carrier is externally chemically identical; (b) attaching a first glycan ligand to one set of silent carriers comprising a first nucleic acid code to form a first set of carriers; (c) repeating step (b) to produce N sets, where N>2, wherein each set comprises a different glycan ligand, or a different density of glycan ligand, and each set comprises a different nucleic acid code; (d) pooling the N sets to form a first mixed library comprising a liquid glycan array; (e) contacting the first mixed library with the target molecule and identifying the set of ligands {M} which bind to the target molecule; (f) repeatedly creating a pooled set of binding glycans, omitting one binding glycan or one density of binding glycan, to form different mixed libraries, and contacting each mixed library with the target model; and (g) determining, using the different nucleic acid codes, which binding glycans have lesser or greater affinity for the target molecule in the absence of the omitted glycan.
2. 2 . The method of claim 1 wherein the carrier is a virus or phage and the target molecule is a lectin.
3. 3 . The method of claim 2 , wherein the plurality of nucleic acid codes comprises degenerate DNA sequences of a portion of a viral or phage protein.
4. 4 . The method of claim 1 , wherein at least one nucleic acid code encodes a unique fluorescent or enzymatic detection marker.
5. 5 . The method of claim 1 wherein a set of silent carriers comprises carriers chemically modified to display a glycan ligand on the surface of the carrier at a specific density.
6. 6 . The method of claim 1 , wherein the identification of binding ligands is performed by extracting nucleic acids from carrier comprising the ligand bound to the target, and amplifying and sequencing the nucleic acids.
7. 7 . The method of claim 6 , wherein a quantitative assessment of the binding of the ligands is assessed by copy number following PCR.
8. 8 . The method of claim 4 wherein the identification of binding ligands is performed by detecting the fluorescent or enzymatic detection marker.
9. 9 . The method of claim 1 , wherein the target molecule is a protein, purified biomolecule, cell, organ, or inorganic material.
10. 10 . The method of claim 1 wherein the identification of binding ligands comprises a step of separating target molecule-ligand-silent carrier complexes in a pull-down assay.
11. 11 . The method of claim 10 wherein the pull down assay comprises a step of binding to a solid support, precipitation, centrifugation, magnetic capture, or partitioning into another solvent.
12. 12 . The method of claim 1 wherein the first mixed library is a liquid mixed library and the target molecule is comprised in a liquid, which target molecule is converted to solid form and separated from the liquid mixture together with ligands which bind to the target molecule.
13. 13 . The method of claim 12 wherein the target molecule is in solution, dispersion, emulsion in the liquid, or is a liquid itself.
14. 14 . The method of claim 13 wherein the target molecule is a salt which is precipitated from solution.
15. 15 . The method of claim 13 wherein the target molecules are aggregated into an insoluble particle.
16. 16 . The method of claim 13 wherein the target molecules are converted from liquid phase to solid phase.
17. 17 . The method of claim 4 wherein the detection marker comprises a reporter protein encoded into the DNA of the carrier such that the detection marker is expressed by a host organism upon infection by carrier.
18. 18 . The method of claim 17 wherein the reporter protein comprises galactosidase, chloramphenicol acetyltransferase, or a fluorescent protein.
19. 19 . A method of claim 1 where a ligand is attached to a carrier by forming a covalent amide bond with lysine or amino terminus of a carrier coat protein.
20. 20 . The method of claim 19 where the carrier coat protein is modified to introduce a reactive handle which is reactive with a cognate reactive handle on the ligand, which cognate reactive handle is not reactive with any other functional group on the coat protein.

Description

FIELD The present application pertains to the field of receptor-ligand interactions and molecular recognition. More particularly, the present application relates to methods for the discovery of ligands or combination of ligands that bind in synergy to a biomolecule of interest. SEQUENCE LISTING A Sequence Listing is provided herewith as an ASCII.txt file “88466.14 Sequence Listing Project_ST25.txt” created on Sep. 4, 2025 and having a size of 10.3 KB. The contents of the Sequence Listing.txt file are incorporated by reference herein in their entirety. BACKGROUND It is known that many proteins and other macromolecular receptors can interact with more than one ligand. Simultaneous interaction of the receptor with two ligands often produces different biophysical, biochemical and physiological outcomes than the interaction of the same receptor with either of the individual ligands. Such interactions, when the binding of two molecules proves to be more advantageous than the binding of either ligand individually, are termed “synergistic” or “positively cooperative” 1. These “synergistic” interactions may be of great interest in fields that deal with receptor-ligand interactions (drug discovery, diagnostics, and basic research). One specific example of a synergistic interaction is that of carbohydrates and proteins. Examples are known where two distinct types of glycans bind to one protein with significantly higher affinity than either one of the glycans alone2-4. Among possible factors, the biophysical origin of such an enhancement may be due to allosteric conformational change within the protein structure or interactions of two molecules. Many known methods in ligand discovery are optimized for discovery of individual ligands that bind to individual proteins2, referred to here as “spatially-separated libraries”. Examples include the screening of libraries of individual molecules on microtiter plates, the screening of molecular arrays, in which each molecule is attached to the surface in a specific location, or the screening of a one-bead-one-compound library where individual macroscopic (micron-sized) beads bear a unique molecule. Upgrading “spatially-separated libraries” technology to permit for screening of synergistic interactions is theoretically possible but, in practice, it can be exponentially more complex. A library of N different molecules contains about N2/2 unique binary combinations. Therefore, for even a small library of 1000 molecules, one needs to produce and test 500,000 binary combinations. This number scales to 200,000,000 for a trinary combination. Thus, to achieve a feasible result, it may be necessary to compromise the complexity of the library (i.e., make the number of tested library members smaller). A well-known technology complementary to “spatially-separated libraries” is a “mixed library” technology, in which multiple molecules are present in the same solution. This technology allows screening of a mixture of molecules and is a “display” technology. In a display technology, each molecule is attached covalently or non-covalently to a nanoscale information-bearing tag, such as DNA, RNA, ribosome, or particle of bacteriophage or virus. A variant of such technology is a SELEX (systematic evolution of ligands by exponential enrichment) or analogous procedure for development of RNA or DNA aptamers, where the encoding entity is the DNA or RNA molecule. However, DNA or RNA can have potential interaction with the receptor, which interaction may be wanted or unwanted. These problems are minimized in phage display technology where the different molecules are immobilized on virus or bacteriophage particles of identical composition and DNA or RNA of different composition is contained within the viral capsid of the phage particle. A mixed library technology is suited for identification of synergistic binding because all molecules are present in the same solution. Identification of synergistic interactions using mixed encoded libraries, however, has not been documented. Several requirements are not obvious: (1) To analyze the synergistic binding, it should be possible to produce a library of N defined components and a nearly identical library with N-m components in which m specific members of the original library are excluded (m<N). (2) A production and application of the mixed library technology has to permit two or more molecules to interact with the same target. For example, Lerner and Brenner, Lam and coworkers, and others teach production of mixed molecular libraries displayed, along with encoding tags, on macroscopic carriers, such as beads of >1 micron in size made of agarose, polystyrene. The size of the carrier bead effectively precludes simultaneous binding of distinct molecules attached to two distinct beads to one protein target of size of <0.01 micron. One technology for the generation of display libraries on nanoscale carriers of identical composition utilizes a recombinant protein tech