Display of molecules on silently genetically encoded nanoscale carriers for determining synergistic molecular interactions
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.
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 . The method of claim 1 wherein the carrier is a virus or phage and the target molecule is a lectin.
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 . The method of claim 1 , wherein at least one nucleic acid code encodes a unique fluorescent or enzymatic detection marker.
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 . 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 . The method of claim 6 , wherein a quantitative assessment of the binding of the ligands is assessed by copy number following PCR.
8 . The method of claim 4 wherein the identification of binding ligands is performed by detecting the fluorescent or enzymatic detection marker.
9 . The method of claim 1 , wherein the target molecule is a protein, purified biomolecule, cell, organ, or inorganic material.
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 . 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 . 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 . The method of claim 12 wherein the target molecule is in solution, dispersion, emulsion in the liquid, or is a liquid itself.
14 . The method of claim 13 wherein the target molecule is a salt which is precipitated from solution.
15 . The method of claim 13 wherein the target molecules are aggregated into an insoluble particle.
16 . The method of claim 13 wherein the target molecules are converted from liquid phase to solid phase.
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 . The method of claim 17 wherein the reporter protein comprises galactosidase, chloramphenicol acetyltransferase, or a fluorescent protein.
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 . 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.