Barcoded Protein Array for Multiplex Single-Molecule Interaction Profiling

Abstract

Methods for attaching barcodes to polypeptides are provided. Methods for detecting molecular interactions at the single molecule level are provided. Embodiments of the invention are directed to a ONA barcoded protein array technology for parallel protein interaction profiling on a single molecule basis. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Novel methods are described herein that measure protein interactions based on the statistical analysis of co-localized polonies arising from barcoding DNAs of interacting proteins.

What is claimed is: 
     
         1 . A method for attaching a plurality of barcodes to a plurality of polypeptides comprising the steps of:
 attaching a barcode to a plurality of DNA template sequences to produce a plurality of barcoded templates comprising a barcode sequence and a protein coding sequence;   performing reverse transcription of the barcoded templates to produce a plurality of mRNA-cDNA hybrid sequences; and   performing in vitro translation of the mRNA-cDNA hybrid sequences to generate a plurality of protein-ribosome-mRNA-cDNA complexes.   
     
     
         2 . The method of  claim 1 , wherein the step of attaching is performed using PCR and in vitro transcription. 
     
     
         3 . The method of  claim 1 , wherein the plurality of protein-ribosome-mRNA-cDNA complexes are formed by in vitro translation and ribosome stalling. 
     
     
         4 . The method of  claim 1 , wherein the plurality of barcoded sequences are synthesized in parallel on an immobilized support or individually synthesized as a mixture of random sequences on a support. 
     
     
         5 . The method of  claim 1 , wherein each of the steps is performed in a single container, and a correlation between a barcoding sequence and a protein sequence is determined using massively parallel DNA sequencing. 
     
     
         6 . The method of  claim 1 , wherein the barcoded templates contain a polymerase promoter, and mRNAs are synthesized from the barcoded DNA templates by in vitro transcription in a single container. 
     
     
         7 . The method of  claim 6 , wherein the polymerase is T7 polymerase. 
     
     
         8 . The method of  claim 1 , wherein the reverse transcription is performed using universal primers, and the cDNA sequences are complementary upstream to a ribosome binding site of the barcoded template. 
     
     
         9 . The method of  claim 1 , wherein ribosomes stall at the 3′ end of the mRNA-cDNA hybrid sequences during in vitro translation due to one or both of a lack of stop codons or the presence of ribosome stalling peptide sequences. 
     
     
         10 . The method of  claim 1 , wherein primers for cDNA synthesis contain one or both of 5′ desthiobiotin modifications and 5′ acrydite modifications. 
     
     
         11 . The method of  claim 1 , wherein the protein coding sequence encodes one or more affinity tags at its C-terminus. 
     
     
         12 . The method of  claim 11 , wherein the affinity tag is a FLAG tag. 
     
     
         13 . The method of  claim 1 , wherein the protein-ribosome-mRNA-cDNA complexes are purified using a protein affinity tag and a cDNA desthiobiotin tag. 
     
     
         14 . A method for attaching a barcode to a polypeptide comprising the steps of:
 providing a DNA template comprising an enzyme ligand at its 5′ end;   providing a protein comprising an enzyme specific for the ligand; and   allowing the enzyme to bind the ligand to produce a polypeptide comprising a barcode sequence.   
     
     
         15 . The method of  claim 14 , performed using an automated high-throughput platform. 
     
     
         16 . The method of  claim 15 , wherein 10,000 or more polypeptides comprising a barcode sequence are prepared in parallel. 
     
     
         17 . The method of  claim 14 , wherein an enzyme ligand is selected from the group consisting of one or more of HaloTag, CLIP tag and SNAP tag. 
     
     
         18 . The method of  claim 14 , wherein both the DNA template and the polypeptide comprise an affinity tag. 
     
     
         19 . The method of  claim 18 , further comprising the step of performing affinity purification using two steps. 
     
     
         20 . The method of  claim 19 , wherein the DNA template comprises a desthiobiotin tag and the polypeptide comprises a His tag. 
     
     
         21 . A method of detecting and quantifying a plurality of polypeptides in situ comprising the steps of:
 providing in an aqueous medium a plurality of polypeptides comprising a barcode;   immobilizing the plurality of polypeptides on a substrate;   performing in situ amplification of the barcodes bound to the immobilized plurality of polypeptides; and   identifying and quantifying amplified barcode sequences and recording their locations by in situ DNA sequencing.   
     
     
         22 . The method of  claim 21 , wherein the polypeptides comprising barcodes are made according to the method of  claim 1  or  claim 14 . 
     
     
         23 . The method of  claim 21 , wherein the plurality of polypeptides are randomly immobilized in a crosslinked polyacrylamide gel layer having a thickness of about a few microns. 
     
     
         24 . The method of  claim 21 , wherein the nucleic acid sequences have a 5′ end modification and are copolymerized into the gel matrix to avoid template drifting. 
     
     
         25 . The method of  claim 24 , wherein the 5′ end modification is an acrydite modification. 
     
     
         26 . The method of  claim 21 , wherein the nucleic acid sequences are amplified into polonies using solid-phase PCR. 
     
     
         27 . The method of  claim 26 , wherein the polonies are approximately 1-2 microns in diameter. 
     
     
         28 . The method of  claim 27 , wherein greater than about 1,000,000 polonies are analyzed on 1 mm2 array area. 
     
     
         29 . The method of  claim 27 , wherein the polonies are analyzed using sequencing-by-synthesis or sequencing-by-ligation to identify barcode sequences and location coordinates. 
     
     
         30 . A method of detecting a protein-protein interaction between two or more polypeptides comprising the steps of:
 providing in an aqueous medium a plurality of polypeptides comprising a barcode under defined conditions to allow formation of protein-protein interactions;   stabilizing the protein-protein interactions by chemical crosslinking;   immobilizing the plurality of polypeptides on a substrate;   performing in situ amplification of the barcodes bound to the immobilized plurality of polypeptides; and   detecting amplified barcode sequences, wherein co-localized amplified barcode sequences are detected when a protein-protein interaction has occurred between two or more polypeptides.   
     
     
         31 . The method of  claim 30 , wherein the polypeptides comprising barcodes are made according to the method of  claim 1  or  claim 14 . 
     
     
         32 . The method of  claim 30 , wherein defined conditions are selected from the group consisting of one or any combination of ligands, cofactors, buffers and temperature. 
     
     
         33 . The method of  claim 30 , wherein co-localized barcodes are deconvoluted by DNA sequencing using the same or different sequencing primers. 
     
     
         34 . The method of  claim 30 , wherein the degree of co-localization of polonies is quantitatively analyzed by co-localization statistics using polony colocalization ratios and pair cross-correlation function (PCCF). 
     
     
         35 . The method of  claim 30 , wherein protein binding affinity can be quantitatively correlated with polony co-localization ratios. 
     
     
         36 . The method of  claim 30 , wherein the polypeptide is selected from the group consisting of a natural polypeptide, a recombinant polypeptide, and a de novo synthesized polypeptide. 
     
     
         37 . The method of  claim 30 , wherein at least about 1,000,000,000 polypeptides are immobilized on half the area of a standard microscopic slide. 
     
     
         38 . The method of  claim 37 , wherein the microscopic slide is 25×75 mm2. 
     
     
         39 . The method of  claim 30 , wherein a first library of at least 100,000 different polypeptides can be screened against a second other library of at least 100,000 different polypeptides or other barcoded molecules in a single assay. 
     
     
         40 . The method of  claim 30 , wherein both molecular binding affinity and specificity can analyzed in a single assay. 
     
     
         41 . A method of detecting an interaction between polypeptides and nucleic acid sequences comprising the steps of:
 providing in an aqueous medium a plurality of polypeptides and nucleic acid sequences comprising a barcode under defined conditions to allow formation of polypeptides-nucleic acid interactions;   stabilizing polypeptide-nucleic acid sequence interactions by chemical crosslinking;   immobilizing polypeptides and nucleic acid sequences on a substrate;   performing in situ amplification of the barcodes bound to the immobilized polypeptides and nucleic acids; and   detecting amplified barcode sequences, wherein co-localized amplified barcode sequences are detected when polypeptide-nucleic acid sequence interactions have occurred between polypeptides and nucleic acid sequences.   
     
     
         42 . A method of detecting an interaction between polypeptides and small molecules comprising the steps of:
 providing in an aqueous medium a plurality of polypeptides and small molecules comprising a barcode under defined conditions to allow formation of polypeptide-small molecule interactions;   stabilizing polypeptide-small molecule interactions by chemical crosslinking;   immobilizing polypeptides and small molecules on a substrate;   performing in situ amplification of the barcodes bound to the immobilized polypeptides and small molecules; and   detecting amplified barcode sequences, wherein co-localized amplified barcode sequences are detected when polypeptide-small molecule interactions have occurred between polypeptides and small molecules.   
     
     
         43 . A method of detecting binding activity changes of a plurality of polypeptides triggered by binding to an unlabeled ligand in solution comprising the steps of:
 providing in an aqueous medium a plurality of polypeptides comprising a barcode;   providing in the aqueous medium a substrate comprising a barcode, wherein the substrate exhibits altered binding affinity to the polypeptides when bound by a ligand; and   quantifying the co-localization of barcodes determine protein and ligand interactions.   
     
     
         44 . The method of  claim 43 , wherein the polypeptides comprising a barcode are made according to the method of  claim 1  or  claim 14 . 
     
     
         45 . The method of  claim 43 , wherein the barcoded substrate is a protein. 
     
     
         46 . The method of  claim 43 , wherein the ligand increases or decreases binding affinity of a polypeptide to a substrate. 
     
     
         47 . The method of  claim 43 , wherein a barcode is associated with a ligand assayed in a well. 
     
     
         48 . The method of  claim 43 , wherein the ligand is an unlabelled small molecule or a protein. 
     
     
         49 . The method of  claim 48 , wherein the protein is selected from the group consisting of an antibody, a nanobody, adnectin, an affibody and DARPin. 
     
     
         50 . The method of  claim 43 , wherein upon polypeptide binding to a ligand, the polypeptide participates in a protein-protein interaction. 
     
     
         51 . The method of  claim 43 , wherein a library of unlabelled ligands are assayed with a polypeptide library in multi-well plate for automatic high-through screening, and wherein, in each well, one ligand is profiled using a polypeptide library. 
     
     
         52 . The method of  claim 51 , wherein both the polypeptide screening and ligand profiling are performed at the same time to minimize assay time. 
     
     
         53 . The method of  claim 43 , wherein mixed proteins at approximately a zeptomole amount are analyzed in a picoliter reactor to minimize reagent costs. 
     
     
         54 . A method of detecting binding affinity of a polypeptide to a compound comprising the steps of:
 providing a plurality of polypeptides having a barcode bound thereto;   contacting the plurality of polypeptides with one or more test compounds;   performing in situ amplification of the barcodes bound to the plurality of polypeptides; and   detecting amplified barcode sequences, wherein co-localized amplified barcode sequences are detected when a polypeptide has bound to a compound, and wherein the number of co-localized amplified barcode sequences relative to non-co-localized amplified barcode sequences correlates with binding affinity to the compound.   
     
     
         55 . The method of  claim 54 , wherein the compound increases or decreases binding affinity of a polypeptide to a substrate. 
     
     
         56 . The method of  claim 54 , wherein the compound modulates one or more activities of the polypeptide. 
     
     
         57 . The method of  claim 54 , wherein the compound is a small molecule, an antibody or a polypeptide. 
     
     
         58 . The method of  claim 57 , wherein upon polypeptide binding to a small molecule, the polypeptide participates in a protein-protein interaction. 
     
     
         59 . A method of detecting binding affinity of a polypeptide to a compound comprising the steps of:
 providing in an aqueous medium a plurality of polypeptides having a barcode bound thereto;   contacting the medium with one or more test compounds;   immobilizing the plurality of polypeptides on a substrate;   performing in situ amplification of the barcodes bound to the immobilized plurality of polypeptides; and   detecting amplified barcodes, wherein co-localized amplified barcodes are detected when a polypeptide has bound to a compound, and wherein the number of co-localized amplified barcodes relative to non-co-localized amplified barcodes correlates with binding affinity to the compound.   
     
     
         60 . A method of screening for a test compound that modulates an activity of a polypeptide comprising the steps of:
 providing a plurality of polypeptides having a barcode bound thereto;   contacting the plurality of polypeptides with one or more test compounds, wherein polypeptide binding to a test compound alters the ability of the polypeptide to participate in a protein-protein interaction;   performing in situ amplification of the barcode sequences bound to the plurality of polypeptides; and   detecting amplified barcode sequences, wherein altered co-localization of amplified barcode sequences in the presence of the test compound is observed when the test compound modulates an activity of the polypeptide.   
     
     
         61 . The method of  claim 60 , wherein test compound binding to a polypeptide modulates the ability of the polypeptide to participate in a protein-protein interaction. 
     
     
         62 . A method of screening for a test compound that modulates an activity of a polypeptide comprising the steps of:
 providing in an aqueous medium a plurality of polypeptides having a barcode sequence bound thereto;   contacting the medium with one or more test compounds, wherein polypeptide binding to a test compound alters the ability of the polypeptide to participate in a protein-protein interaction;   immobilizing the plurality of polypeptides on a substrate;   performing in situ amplification of the barcode sequences bound to the immobilized plurality of polypeptides; and   
       detecting amplified barcode sequences, wherein altered co-localization of amplified barcode sequences in the presence of the test compound is observed when the test compound modulates an activity of the polypeptide.