Patent Application
20210254073
The present application relates to methods for capture and analysis of macromolecular complexes.
Characterizing macromolecular complexes (MCs) and their interactions is essential for understanding any biological process at the molecular level. With increased resolution and throughput of mass spectrometry (MS) in the last decade, MS-based proteomic analyses following co-immunoprecipitations have been extensively utilized to identify interacting partners of many proteins (DeBlasio et al., “Insights into the Polerovirus-plant Interactome Revealed by Coimmunoprecipitation and Mass Spectrometry,” Mol. Plant Microbe Interact. 28:467-481 (2015); Budayeva et al., “A Mass Spectrometry View of Stable and Transient Protein Interactions,” Adv. Exp. Med. Biol. 806:263-282 (2014); Bauer et al., “Affinity Purification-mass Spectrometry. Powerful Tools for the Characterization of Protein Complexes,” Eur. J. Biochem. 270: 570-578 (2003)). These assays rely on the availability of antibodies that are well characterized, highly specific, and high-affinity against the protein of interest (POI) or a peptide tag that allow the binding partners of the (tagged)-target protein to be co-precipitated while the antibody is immobilized on beads/resins. Even for a single antibody, significant lot-to-lot variability affects the purity, specificity and yield of (co-) immunoprecipitations. The (co-) immunoprecipitated proteins are subsequently eluted by denaturation (i.e. with heat, SDS or combinations) or by on-bead proteolytic digestion and analyzed by MS. However, contaminating peptides derived from the antibody/serum or Protein-A/G are routinely found at an order of magnitude higher abundance than the POI (DeBlasio et al., “Insights into the Polerovirus-plant Interactome Revealed by Coimmunoprecipitation and Mass Spectrometry,” Mol. Plant Microbe Interact. 28:467-481 (2015); Budayeva et al., “A Mass Spectrometry View of Stable and Transient Protein Interactions,” Adv. Exp. Med. Biol., 806:263-282 (2014)). This can hinder the identification of interacting partners, particularly if they are rare or substoichiometric. In some cases, eluates are further fractionated by gel electrophoresis and individual protein bands are excised to exclude heavy and light chains of the antibody prior to MS. This method limits the number of proteins that can be identified and prevents analysis of proteins below the limit of detection for a given electrophoresis/protein staining technique (Jafari et al., “Comparison of In-gel Protein Separation Techniques Commonly Used for Fractionation in Mass Spectrometry-based Proteomic Profiling,” Electrophoresis 33:2516-2526 (2012)) while increasing the likelihood of keratin contamination and the length of time needed for sample preparation.
The present application is directed to overcoming these and other deficiencies in the art.
The present application relates to a method for analyzing a molecular target in a sample. The method involves providing an aptamer, wherein the aptamer is a high affinity binding partner to at least a portion of the molecular target in the sample. The aptamer is contacted with the sample containing the molecular target under conditions effective for the molecular target and aptamer to bind to each other. The molecular target is separated from the sample to form a molecular target enriched sample. The separated molecular target of the enriched sample is then analyzed.
In some embodiments, the separated molecular target is analyzed using mass spectrometry. In some embodiments, the separated molecular target is analyzed using cryo-electron microscopy. In some embodiments, the separated molecular target is analyzed using nucleotide sequencing. In some embodiments, the separated molecular target is analyzed using any combination of these techniques.
To provide an alternative to immunoprecipitations, an RNA aptamer-based affinity purification method has been developed using the highly-specific and high-affinity Green Fluorescent Protein (GFP)-aptamer (Shui et al., “RNA Aptamers that Functionally Interact with Green Fluorescent Protein and its Derivatives,” Nucleic Acids Res. 40:e39 (2012), which is hereby incorporated by reference in its entirety) to co-purify GFP-tagged target proteins and their binding partners for identification by mass spectrometry (MS) (AptA-MS), nucleotide sequencing, and/or cryo-electron microscopy. Nucleic acid aptamers can be selected against a wide variety of targets and synthesized in unlimited quantities by cost-effective methods. These properties, in addition to their high specificity and affinity, make aptamers attractive reagents for affinity purification. Indeed, aptamers have been used for affinity purification of targets from biological mixtures followed by MS, but mainly for target detection and biomarker discovery (Gulbakan, “Oligonucleotide Aptamers: Emerging Affinity Probes for Bioanalytical Mass Spectrometry and Biomarker Discovery,” Anal. Methods 7:7416-7430 (2015), which is hereby incorporated by reference in its entirety). These detection assays were developed with a handful of aptamers and demonstrated to work by proof-of-principle experiments with little or no biological applications. General and simple affinity-capture methods using RNA aptamers are lacking, especially those that allow for quantitative analysis of protein interactions and protein complex formation directly from cellular lysates and can be applied to address a broad array of biological questions in a wide range of species, tissues, and cell types.
The GFP protein in combination with the high-affinity and high-specificity GFP-aptamer (Shui et al., “RNA Aptamers that Functionally Interact with Green Fluorescent Protein and its Derivatives,” Nucleic Acids Res. 40:e39 (2012), which is hereby incorporated by reference in its entirety) serves as a suitable affinity tool to identify, for example, protein-protein interactions by MS, three-dimensional structure by cryo-electron microscopy, and DNA binding sites of proteins within a complex using nucleotide sequencing techniques. The methods described herein allow use with a broad collection of existing GFP-fusion proteins in human cells and other model organisms including Drosophila and yeast. Here, it is demonstrated that AptA-MS is superior to conventional co-immunoprecipitations for subsequent MS analysis, because it is devoid of immunoprecipitation-derived protein contaminants, and provides a dramatic enrichment of the POI. Using AptA-MS several known and novel interactors of human Heat Shock Factor 1 (HSF1) tagged with GFP have been identified, some of which showed an increased association following heat shock (HS). In addition, post-translational modifications (PTMs) of HSF1 and the co-precipitated histones have been identified without additional tailored enrichment steps for these modifications. AptA-MS has also been applied with other aptamers (e.g. NELF-aptamer) (Pagano et al., “Defining NELF-E RNA Binding in HIV-1 and Promoter-proximal Pause Regions,” PLoS Genet. 10:e1004090 (2014), which is hereby incorporated by reference in its entirety) to enrich its target from Drosophila S2 cells. The results indicate that in addition to purifying transiently transfected HSF1-GFP from human cells, the GFP-aptamer is capable of enriching endogenous GFP-tagged RNA polymerase II (Pol II) from yeast, as well as formaldehyde crosslinked GFP from Drosophila S2 cells, thereby making it a versatile tool for affinity purification of GFP-tagged proteins from various sources.
The present application is directed to a method for analyzing a molecular target in a sample. The method involves providing an aptamer, wherein the aptamer is a high affinity binding partner to at least a portion of the molecular target in the sample. The aptamer is contacted with the sample containing the molecular target under conditions effective for the molecular target and aptamer to bind to each other. The molecular target is separated from the sample to form a molecular target enriched sample. The separated molecular target of the enriched sample is then analyzed.
The sample may be any biological sample, including and without limitation, a cell or tissue lysate, a plasma sample, a serum sample, a blood sample, an exosome sample, or other biological sample. In some embodiments, the biological sample is derived from a cell sample, e.g., a cell lysate of a population of cells grown in vitro or in vivo. Suitable cells include immortalized cells of a cell line or primary cells. Cells can be non-mammalian or mammalian cells (e.g., a preparation of rodent cells, rabbit cells, guinea pig cells, feline cells, canine cells, porcine cells, equine cells, bovine cell, ovine cells, monkey cells, or human cells). In some embodiments, the sample is derived from preparation of human cells. Suitable cell samples also include primary or immortalized embryonic cells, fetal cells, or adult cells, at any stage of their lineage, e.g., totipotent, pluripotent, multipotent, or differentiated cells.
The molecular target in the sample that is subject to analysis according to the methods described herein may comprise one or more biomolecules. In some embodiments, the molecular target comprises at least two, at least three, at least four, at least five, or greater than five biomolecules in complex together. In some embodiments, the one or more biomolecules of the molecular target are proteins, polypeptides, peptides, ribonucleic acid molecules (RNA), deoxyribonucleic acid molecules (DNA), lipids, carbohydrates, or any combination thereof.
The molecular target in the sample that is subject to analysis according to the methods described herein may comprise one or more biomolecules that are transiently introduced into a sample (e.g., through transient transfection) or that are endogenously present in the sample. Methods of transient transfection are well known to a person of ordinary skill in the art.
In some embodiments, the sample is crosslinked to maintain the integrity of the molecular target therein. Crosslinking the sample is generally carried out prior to contacting the sample with the aptamer. Methods of crosslinking a sample and/or the components of the sample are well known in the art and include, for example, ultraviolet irradiation, chemical and physical (e.g., optical) crosslinking. Non-limiting examples of chemical crosslinking agents include formaldehyde and psoralen (Solomon et al., Proc. Natl. Acad. Sci. USA 82:6470-6474 (1985); Solomon et al., Cell 53:937947 (1988), which are hereby incorporated by reference in their entirety). Cross-linking is performed using any of a number of approaches known in the art, such as by adding a solution comprising formaldehyde, as described in the Examples herein, to a sample comprising nucleic acid molecules and chromatin proteins. In some embodiments, the sample is crosslinked by exposing the sample to a 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% of >1% solution of formaldehyde. Other non-limiting examples of agents that can be used for cross-linking DNA and proteins include, but are not limited to, mitomycin C, nitrogen mustard, melphalan, 1,3-butadiene diepoxide, cis diaminedichloroplatinum(II) and cyclophosphamide.
In other embodiments, the sample is “native”, or not crosslinked, prior to contacting with the aptamer.
As used herein, the term “aptamer” refers to an oligonucleotide molecule, or peptide molecule that binds to a specific molecular target.
In some embodiments, the aptamer is a peptide aptamer. As described in Reverdatto et al., “Peptide Aptamers; Development and Applications,” Curr Top Med Chem 15(12):1082-1101 (2015), which is hereby incorporated by reference in its entirety, peptide aptamers contemplated for use in the method of the present application are small combinatorial proteins that are selected to bind to specific sites on target molecules. Generally, peptide aptamers consist of 5-20 amino acid long residue sequences and are typically embedded as a loop within a stable protein scaffold.
In other embodiments, the aptamer is an oligonucleotide molecule. Oligonucleotide aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. Aptamers assume a variety of shapes forming helices, single stranded loops, stem loops, etc. that provides a tertiary structure responsible for target molecule binding. Aptamer target binding involves three dimensional, shape dependent interactions as well as hydrophobic interactions, base stacking and intercalation.
In accordance with the methods of the present application, the aptamer may be a nucleic acid, preferably an RNA aptamer. In some embodiments, the aptamer is a non-naturally occurring, artificially-engineered aptamer. In other embodiments, the aptamer is a natural aptamer. In some embodiments, the aptamer utilized in the methods herein is an RNA aptamer comprising at least two stem loops, at least three stem loops, or at least four stem loops. In some embodiments, the aptamer utilized in the methods disclosed herein comprises an internal loop and at least two flanking stems (preferably three stems). In some embodiments, the aptamers are designed and synthesized or randomly synthesized. The aptamers can be screened, selected or ranked by affinity binding test with a molecular target. In other embodiments, the aptamers are amplified from a selected sequence or optimized sequence.
The nucleotide components of aptamers suitable for use in accordance with the methods described herein may include modified or non-natural nucleotides to enhance stability or other desired properties. Accordingly, in some embodiments, aptamers of the disclosure comprise one or more modified sugar groups (e.g., modifications at the 2′ position of the ribose to include a 2′-amino, 2′-fluoro, or 2′O-methyl group) to enhance resistance to nucleases and improved stability. In some embodiments, aptamers of the disclosure comprise a phosphodiester linkage modification, e.g., incorporation of phosphorothioate or boranophosphate linkages, to enhance resistance to nucleases. In some embodiments, the aptamers of the disclosure are made of L-ribose-based nucleotides instead of natural D-nucleotides to confer nuclease resistance. In some embodiments, aptamers of the disclosure are composed of locked nucleic acids or analogues thereof. In some embodiments, the aptamers of the disclosure comprise a 3′ end cap to prevent nuclease resistance.
As described herein the aptamer is a high affinity binding partner to at least a portion of the molecular target in the sample. As used herein, a “high affinity binding partner” generally has a low dissociation constant with its molecular target. In some embodiments, the aptamer has high affinity to a molecular target protein or a molecular tag fused with a molecular target. In some embodiments, the high affinity binding is reversible between the aptamer and the molecular target (or molecular target fused with a tag). In some embodiments, the binding can be dissociated by a reagent with higher affinity to the aptamer or a reagent capable of changing the conformation of the aptamer or the molecular target. The binding affinity can be measured by equilibrium dissociation constant (KD) of the aptamer and the target protein. The smaller the KD value, the greater the binding affinity of the aptamer for its target protein. The larger the KD value, the weaker the binding affinity. In some embodiments, the KD of the aptamer and target protein is less than 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 50 nM, or 100 nM. In some embodiments, the aptamer binds green fluorescent protein tag of the molecular target, and the KD of the aptamer is less than 20 nM, less than 10 nM, less than 5 nM or between 1-50 nM or preferably between 5-20 nM.
In identifying suitable aptamers for use in the methods of the present application, a person of skill in the art would understand that this involves selecting aptamers that bind the molecular target with sufficiently high affinity (e.g., Kd<50 nM) and specificity from a pool of nucleic acids containing a random region of varying or predetermined length (Shi et al., “A Specific RNA Hairpin Loop Structure Binds the RNA Recognition Motifs of the Drosophila SR Protein B52,” Mol. Cell Biol. 17:1649-1657 (1997); Shi, “Perturbing Protein Function with RNA Aptamers,” Thesis, Cornell University, University Microfilms, Inc. (1997), which are hereby incorporated by reference in their entirety).
For example, identifying suitable aptamers of the present application can be carried out using an established in vitro selection and amplification scheme known as SELEX. The SELEX scheme is described in detail in U.S. Pat. No. 5,270,163 to Gold et al.; Ellington and Szostak, “In Vitro Selection of RNA Molecules that Bind Specific Ligands,” Nature 346:818-822 (1990); and Tuerk and Gold, “Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA Polymerase,” Science 249:505-510 (1990), which are hereby incorporated by reference in their entirety. The SELEX procedure can be modified so that an entire pool of aptamers with binding affinity can be identified by selectively partitioning the pool of aptamers. This procedure is described in U.S. Patent Application Publication No. 2004/0053310, which is hereby incorporated by reference in its entirety.
In some embodiments, the portion of the molecular target bound by the aptamer is a tag moiety. In some embodiments, the aptamer is a high affinity binding partner to at least a portion of the tag moiety of the molecular target. The tag moiety can be fused or conjugated to a protein, an RNA, a DNA, a lipid, or a carbohydrate component of the molecular target.
Exemplary tag moieties that can be fused or conjugated to the molecular target in accordance with the methods disclosed herein include, without limitation, a fluorescent protein or fragment thereof, a poly-His tag, a maltose binding protein tag, an albumin-binding protein tag, a calmodulin binding peptide tag, a glutathione S-transferase tag, a chitin binding protein tag, FLAG-tag, HA-tag, Protein A tag, and combinations thereof. Aptamers to various tag moieties described herein and are known in the art. For example, WO 2014/185802 to Bartnicki et al. describes DNA aptamers that bind to His tags which are suitable for use in accordance with the methods of the disclosure, and U.S. Patent Application Publication No. 2010/0036106 to Yoshida et al. describes high affinity RNA aptamers that bind with high affinity to GST tags, which are also suitable for use in accordance with the methods of the disclosure. Fusing, conjugating, or labeling a protein, an RNA, a DNA, a lipid, or a carbohydrate with a tag moiety can be carried out using standard molecular biology techniques that are well known to those of skill in the art
In some embodiments, the molecular target is fused or conjugated to a fluorescent protein tag. Fluorescent protein tags can be a naturally occurring protein or an engineered protein, such as a derivative of the naturally occurring fluorescent protein.
Exemplary fluorescent proteins include, without limitation, Aequorea-derived proteins such as Green Fluorescent Protein (“GFP”), enhanced Green Fluorescent Protein (“eGFP”), Yellow Fluorescent Protein (“YFP”), enhanced Yellow Fluorescent Protein (“EYFP”), and Cyan Fluorescent Protein (“CFP”), Enhanced Cyan Fluorescent Protein (“ECFP”), their variants or the combination thereof, as well as proteins derived from coral species including, but not limited to, Discosoma and Trachyphyllia geoffroyi. Other proteins having fluorescent or other signaling properties that are known in the art and commercially available can also be used.
Exemplary modified fluorescent proteins that can also be utilized as tag moieties in the methods of the present disclosure include those that contain one or more of the following modifications: circular permutation (Baird et al., “Circular Permutation and Receptor Insertion Within Green Fluorescent Proteins,” Proc. Natl. Acad. Sci. USA 96:11241-11246 (1999), which is hereby incorporated by reference in its entirety), splitting (Zhang et al., “Combinatorial Marking of Cells and Organelles with Reconstituted Fluorescent Proteins,” Cell 119:137-144 (2004), which is hereby incorporated by reference in its entirety), enhanced folding (Pedelacq et al., “Engineering and Characterization of a Superfolder Green Fluorescent Protein,” Nat. Biotechnol. 24:79-88 (2006), which is hereby incorporated by reference in its entirety), or other modifications (Zhang et al., “Creating New Fluorescent Probes for Cell Biology,” Nat. Rev. Mol. Cell Biol. 3:906-918 (2002), which is hereby incorporated by reference in its entirety).
Specific examples of fluorescent proteins suitable for use in accordance with the methods disclosed herein (and their encoding nucleic acids) are well known in the art including, without limitation, those reported as Genbank Accessions AB195239, DD431502-DD431504, DD420089-DD420091, AY013821, AY013824-AY013827, EF064258-EF064259, AF435-427-AF435-434, DQ092360-DQ092365, DQ525024-DQ525025, X83959-X83960, AY533296, AB041904, X96418, BD136947-BD136949, U73901, AX250563-AX250571, AF302837, AF183395, AF058694-AF058695, U50963, L29345, M62653-M62654, DQ301560, AY679106-AY679108, AY678264-AY678271, AF168419-AF168420, AF272711, AY786536-AY786537, AF545828, AF506025-AF506027, AF420593, BAC20344, BD440518-BD440519, and AB085641, each of which is hereby incorporated by reference in its entirety.
In some embodiments of the present disclosure, the aptamer is a nucleic acid aptamer that binds to a fluorescent protein, e.g., GFP, eGFP, eCFP, and eYFP. In accordance with these embodiments, the fluorescent protein is a tag moiety associated with the molecular target being analyzed in the method of the present application. Exemplary nucleic acid aptamers that bind fluorescent proteins that are suitable for use in the methods described herein include those disclosed in U.S. Pat. No. 8,445,655 to Kotlikoff et al., and Shui et al., “RNA Aptamers that Functionally Interact with Green Fluorescent Protein and its Derivatives,” Nucleic Acids Res. 40(5): e39 (2012), which are hereby incorporated by reference in their entirety. These nucleic acid aptamers have a core region sequence that is the primary portion of the aptamer having fluorescent protein binding activity. Accordingly, suitable aptamers that bind with high affinity to green fluorescent proteins for use in the methods disclosed herein include aptamers having any one of the core sequences provided in Table 1 below.
The RNA aptamers described above, may further comprise a first primer region located 5′ to the core region, a second primer region located 3′ to the core region; and one or both of a 5′ random nucleotide sequence region and a 3′ random nucleotide sequence region. These nucleic acid aptamers assume a secondary structure comprising three stems and a central loop.
In another embodiment, the aptamer is a nucleic acid aptamer that binds to a fluorescent protein and is based on nucleic acid sequences described in Tome et al., “Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling,” Nat Methods (11): 683-688 (2014), which is hereby incorporated by reference in its entirety. Accordingly, an exemplary nucleic acid aptamer for use in the methods described herein includes the GFP aptamer having the sequence of:
In another embodiment, the nucleic acid aptamer is an aptamer that binds to negative elongation factor (NELF). Suitable nucleic acid aptamers that bind NELF include those disclosed in Tome et al., “Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling,” Nat Methods (11): 683-688 (2014), which is hereby incorporated by reference in its entirety. An exemplary nucleic acid aptamer that binds to NELF for use in the methods described herein has the sequence of:
In accordance with the methods described herein, a suitable aptamer is contacted with the sample containing the molecular target under conditions effective for the molecular target and aptamer to bind to each other. The molecular target bound to the aptamer is then separated from the sample to form a molecular target enriched sample.
In some embodiments, the aptamer (e.g., a nucleic acid aptamer) is immobilized on a solid support. Suitable solid supports for use in the methods of the present application include, without limitation, a bead, a particle, a column, a fluidic device, a microfluidic device, a microarray, a strand, a gel, a sheet, a tube, a sphere, a capillary, a pad, a film, a plate, a disc, and a membrane. The solid support may have any convenient shape, such as a disc, square, circle, etc., and may contain raised or depressed regions suitable for immobilization of aptamers of the present disclosure. By way of example, the aptamer is immobilized on a surface and/or the interior of a bead, substrate, matrix or network via high affinity binding complex. The bead, substrate, matrix or network can be coated with one part of a high affinity binding complex (e.g., streptavidin), and the aptamer is conjugated to the other part of the high affinity binding complex (e.g., biotin). The aptamer and molecular target is then eluted from the solid support. The elution buffer may comprise the other part of the high affinity binding complex.
As noted above, immobilization of the aptamer to a suitable solid support can be achieved by covalent linkage or by noncovalent interaction (e.g., biotinylated DNA bound to avidin coated beads). Accordingly, the surface of the solid support is coated with a first binding partner of a binding complex and the aptamer may be coupled to a second binding partner of the binding complex, and the aptamer is immobilized on the solid support via binding between the first and second binding partners of the binding complex. Suitable first and second binding partners for immobilizing an aptamer to a solid support in accordance with this embodiment of the present application include, without limitation, biotin and streptavidin, desthiobiotin and streptavidin, maltose and maltose binding protein (MBP), chitin and chitin binding protein, amylose and MBP, glutathione and glutathione-S-transferase, and Ni2+-NTA and His-tag, and integrin and integrin binding peptide. Methods of covalently attaching oligonucleotides to a solid support are well known in the art, see e.g., Gosh and Musso, “Covalent Attachment of Oligonucleotides to Solid Supports,” Nucleic Acids Res. 15(13): 5353-5372 (1987), Joos et al., “Covalent Attachment of Hybridizable Oligonucleotides to Glass Supports,” Anal. Biochem. 247(1):96-101 (1997); Lund et al., “Assessment of Methods for Covalent Binding of Nucleic Acids to Magnetic Beads, Dynabeads, and the Characteristics of the Bound Nucleic Acids in Hybridization Reactions,” Nucleic Acids Res. 16(22):10861-80 (1988), which are hereby incorporated by reference in their entirety.
In some embodiments, the aptamer is reusable or can be rejuvenated or regenerated. The aptamer (e.g. immobilized aptamer) can be used for at least 2 runs (or rounds), at least 3, 4, 5, 10 runs (or rounds) for purifying molecular targets.
The molecular targets collected by using aptamer separation or purification is free or substantially free of other non-specific proteins or contaminating proteins without additional purification process to remove the contaminant proteins. The molecular target collected after the aptamer separation forms a molecular target enriched sample. In some embodiments, the molecular target of this enriched sample is subject to analysis as described here. In some embodiments, the method further comprises an additional enrichment step. This enrichment step may be added either before or after binding of the aptamer to the molecular target. Addition of such a step to the method of the present application allows for a sequential affinity-purification to enrich for a particular molecular target of interest, e.g., a molecular target known to comprise two or more biomolecules. This technique is particularly useful when the aptamer bound portion of the molecular target is a known constituent of more than one macromolecular complex, and it is desirable to analyze a particular macromolecular complex. The technique is also useful to simply improve the purity of the molecular target prior to analysis.
One embodiment of the sequential enrichment step is depicted in
Accordingly, the method of the present application may further involve providing, after separating the molecular target from the sample to form a molecular target enriched sample, a binding agent that binds to a different portion of the molecular target than bound by the aptamer. The molecular target enriched sample is contacted with the binding agent under conditions effective for the molecular target and binding agent to bind to each other. The binding agent is then immunoprecipitated, thereby enriching/isolating the bound molecular target.
Alternatively, as depicted in
In accordance with these embodiments, suitable binding agents for use in the sequential enrichments steps of the methods described herein include any binding agent typically used for immunoprecipitation, including, without limitation, full antibodies, epitope binding fragments of whole antibodies, antibody derivative, antibody mimics, aptamers, etc.
In some embodiments, the binding agent is a full antibody, composed of two light chains and two heavy chain, where the variable regions of each chain (i.e., VL and VH) form the epitope binding region of the antibody. In some embodiments, the binding agent is an epitope binding fragment of an antibody. Examples of the epitope-binding fragments that can be utilized in the methods described herein include Fab′ or Fab fragments (i.e., monovalent fragments containing the VL, VH, CL and CH1 domains); F(ab′)2 fragments (i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments (consisting essentially of the VH and CH1 domains); Fv fragments consisting essentially of a VL and VH domain; and dAb fragments, which consist essentially of a VH or VL domain. In some embodiments, the binding agent is an antibody derivative, which is a molecule that contains at least one epitope binding domain of any antibody. An exemplary antibody derivative for use in the methods described herein is a single chain Fv (“scFv”) antibody, which comprises covalently linked VH::VL heterodimer to form a single antigen binding domain.
When used to sequentially enrich the molecular target as described herein, the antibody, binding fragment thereof, or derivative thereof binds to portion of the molecular target that is different than the portion bound by the aptamer. This portion may be a structural or sequence portion of the molecular target itself or another tag moiety as described infra. For example, as shown in
Once the molecular target has been sufficiently separated from the sample, it is analyzed using any one of a variety of methods well known in the art including, without limitation, mass spectrometry, cryo-electron microscopy, and nucleotide sequencing.
In some embodiments, the molecular target comprises two or more associated biomolecules and analysis of these associated biomolecules involves identifying each of the two or more associated biomolecules of the molecular target.
In accordance with this embodiment, the separated or immunoprecipitated molecular target may be subjected to mass spectrometry to detect each of the two or more associated biomolecules. The use of mass spectrometry for identification of associated biomolecules is well known in the art and described in, for example, Yugandhar et al., “Inferring Protein-Protein Interaction Networks From Mass Spectrometry-Based Proteomic Approaches: a Mini-Review,” Comput Struct Biotechnol J 17:805-811 (2019), which is hereby incorporated by reference in its entirety.
In some embodiments, the molecular target is directly analyzed using MS without the incorporation of any additional separation process to remove contaminant proteins. In some embodiments, the molecular target is subject to one or more additional purification steps to remove contaminant proteins.
Identification of low-abundant proteins by MS becomes possible due to high specificity and enrichment by the aptamer. This also helps in identifying post-translational modifications of the captured proteins without the need for any modification-specific enrichment step. Thus, in some embodiments, the separated or immunoprecipitated molecular target of the method described herein is subjected to mass spectrometry to detect one or more posttranslational modifications of the molecular target. Posttranslational modifications of the molecular target that can be detected using MS include, without limitation, methylation, acetylation, phosphorylation, sumoylation, or combinations thereof. The presence of covalent modifications in proteins affects the molecular weight of the modified amino acids, and the mass increment or deficit can be detected by MS. Accordingly, when practicing the methods of the present application, MS analysis of the molecular target can be used for very high sensitivity detection of post translational modifications in the molecular target, to identify the site of the post translational modification within the molecular target, discover the presence of novel post translational modifications in the molecular target and associated protein sand finally to quantify the relative changes in post translational modification occupancy at distinct sites in the molecular target (Larsen et al., “Analysis of Posttranslational Modification of Proteins by Tandem Mass Spectrometry,” Biotechniques 40(6):790-797 (2018), which is hereby incorporated by reference in its entirety). Methods for identification of post-translational modifications by MS are described in, for example, Larsen et al., “Analysis of Posttranslational Modification of Proteins by Tandem Mass Spectrometry,” Biotechniques 40(6):790-797 (2018), which is hereby incorporated by reference in its entirety.
In some embodiments, analysis of the molecular target is performed using cryo-electron microscopy. The separated or immunoprecipitated molecular target is subjected to cryo-electron microscopy to determine the three-dimensional structure of the molecular target. In some embodiments, the three-dimensional structure of two or more associated biomolecules is determined.
In electron cryo-microscopy (cryoEM), the purified molecular target (e.g., target protein or protein complex) is preserved in vitreous water on sample grids allowing for its native structural state to be maintained (Nogales et al., “Cryo-EM:a Unique Tool for the Visualization of Macromolecular Complexity,” Mol. Cell 58:677-689 (2015), which is hereby incorporated by reference in its entirety). The use of cryoEM in analysis of molecular targets is known in the art and reviewed in, for example, Murata et al., “Cryo-electron Microscopy for Structural Analysis of Dynamic Biological Macromolecules,” Biochimica et Biophysica Acta 1862(2):3240334 (2018), which is hereby incorporated by reference in its entirety.
In some embodiments, the molecular target comprises nucleotide oligomers and the analysis involves isolating the nucleotide oligomers from the immunoprecipitated or separated molecular target and subjecting the isolated nucleotide oligomers to an amplification reaction, a sequencing reaction, or a combination thereof to identify the isolated nucleotide oligomers of the molecular target.
Amplification of nucleotide oligomers that have been isolated from the immunoprecipitated or separated molecular target may be performed using nucleic acid amplification methods well known in the art. These methods include, without limitation, polymerase chain reaction (PCR) (U.S. Pat. No. 5,219,727, which is hereby incorporated by reference in its entirety) and its variants such as in situ polymerase chain reaction (U.S. Pat. No. 5,538,871, which is hereby incorporated by reference in its entirety), quantitative polymerase chain reaction (U.S. Pat. No. 5,219,727, which is hereby incorporated by reference in its entirety), nested polymerase chain reaction (U.S. Pat. No. 5,556,773), self-sustained sequence replication and its variants (Guatelli et al. “Isothermal, In vitro Amplification of Nucleic Acids by a Multienzyme Reaction Modeled after Retroviral Replication,” Proc Natl Acad Sci USA 87(5): 1874-8 (1990), which is hereby incorporated by reference in its entirety), transcriptional amplification and its variants (Kwoh et al. “Transcription-based Amplification System and Detection of Amplified Human Immunodeficiency Virus type 1 with a Bead-Based Sandwich Hybridization Format,” Proc Natl Acad Sci USA 86(4): 1173-7 (1989), which is hereby incorporated by reference in its entirety), Qb Replicase and its variants (Miele et al. “Autocatalytic Replication of a Recombinant RNA.” J Mol Biol 171(3): 281-95 (1983), which is hereby incorporated by reference in its entirety), cold-PCR (Li et al. “Replacing PCR with COLD-PCR Enriches Variant DNA Sequences and Redefines the Sensitivity of Genetic Testing.” Nat Med 14(5): 579-84 (2008), which is hereby incorporated by reference in its entirety) or any other nucleic acid amplification method known in the art. Depending on the amplification technique that is employed, the amplified molecules are detected during amplification (e.g., real-time PCR) or subsequent to amplification using detection techniques known to those of skill in the art. Suitable nucleic acid detection assays include, for example and without limitation, northern blot, microarray, serial analysis of gene expression (SAGE), next-generation RNA sequencing (e.g., deep sequencing, whole transcriptome sequencing, exome sequencing), gene expression analysis by massively parallel signature sequencing (MPSS), immune-derived colorimetric assays, and mass spectrometry (MS) methods (e.g., MassARRAY® System).
Alternatively, sequencing of nucleotide oligomers that have been isolated from the immunoprecipitated or separated molecular target may be performed using methods of nucleotide sequencing that are well known in the art. Illustrative non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing, as well as “next generation” sequencing techniques. Those of ordinary skill in the art will recognize that because RNA is less stable in the cell and more prone to nuclease attack experimentally RNA is usually, although not necessarily, reverse transcribed to DNA before sequencing.
Additional DNA sequencing techniques suitable for use in the analysis of the molecular target that are known in the art include, without limitation, fluorescence-based sequencing methodologies (See, e.g., Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y.; which is hereby by reference in its entirety). In some embodiments, automated sequencing techniques are utilized, or the systems, devices, and methods employ parallel sequencing of partitioned amplicons (PCT Publication No: WO2006084132 to Kevin McKernan et al., which is hereby incorporated by reference in its entirety). DNA sequencing may also be achieved by parallel oligonucleotide extension (See, e.g., U.S. Pat. No. 5,750,341 to Macevicz et al., and U.S. Pat. No. 6,306,597 to Macevicz et al., both of which are hereby incorporated by reference in their entireties). Additional examples of sequencing techniques include the Church polony technology (Mitra et al., Analytical Biochemistry 320, 55-65 (2003); Shendure et al., Science 309, 1728-1732 (2005); U.S. Pat. Nos. 6,432,360, 6,485,944, U.S. Pat. No. 6,511,803; which are hereby incorporated by reference in their entireties) the 454 picotiter pyrosequencing technology (Margulies et al., Nature 437, 376-380 (2005); US 20050130173; herein incorporated by reference in their entireties), the Solexa single base addition technology (Bennett et al., Pharmacogenomics 6:373-382 (2005); U.S. Pat. Nos. 6,787,308; 6,833,246; which are hereby incorporated by reference in their entirety), Illumina Single base sequencing technology, the Lynx massively parallel signature sequencing technology (Brenner et al. Nat. Biotechnol. 18:630-634 (2000); U.S. Pat. Nos. 5,695,934; 5,714,330; which are hereby incorporated by reference in their entirety) and the Adessi PCR colony technology (Adessi et al. Nucleic Acid Res. 28, E87 (2000); WO 00018957, which are hereby incorporated by reference in their entirety).
A set of methods referred to as “next-generation sequencing” techniques (Voelkerding et al., Clinical Chem. 55: 641-658 (2009); MacLean et al., Nature Rev. Microbiol., 7: 287-296, which are hereby incorporated by reference in their entirety) may also be used in accordance with this embodiment. These techniques may be used to sequence the nucleotide oligomers that have been isolated from the immunoprecipitated or separated molecular target. Next-generation sequencing (NGS) methods share the common feature of massively parallel, high-throughput strategies, with the goal of lower costs in comparison to older sequencing methods. NGS methods can be broadly divided into those that require template amplification and those that do not. Amplification-requiring methods include pyrosequencing commercialized by Roche as the 454 technology platforms (e.g., GS 20 and GS FLX), the Solexa platform commercialized by Illumina, and the Supported Oligonucleotide Ligation and Detection (SOLiD) platform commercialized by Applied Biosystems (Voelkerding et al., Clinical Chem. 55: 641-658 (2009); MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No. 5,912,148; U.S. Pat. No. 6,130,073; which are hereby incorporated by reference in their entirety). Non-amplification approaches, also known as single-molecule sequencing, are exemplified by the HeliScope platform commercialized by Helicos BioSciences, and emerging platforms commercialized by VisiGen, Oxford Nanopore Technologies Ltd., and Pacific Biosciences, respectively.
Other emerging single molecule sequencing methods useful in the methods of the present application include real-time sequencing by synthesis using a VisiGen platform (Voelkerding et al., Clinical Chem. 55: 641-658 (2009); U.S. Pat. No. 7,329,492; U.S. patent application Ser. No. 11/671,956; U.S. patent application Ser. No. 11/781,166; which are hereby incorporated by reference in their entirety).
While certain embodiments have been set forth, alternative embodiments and various modifications will be apparent form the above description to those skilled in the art. These and other alternatives are considered equivalents and within the spirit and scope of this disclosure.
The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof.
Cell culture and transfection. Human HCT116 cells were grown in McCoy's 5A media (with 10% FBS+P/S) at 37° C. Around 4 million HCT116 cells were plated in McCoy's 5A media (with 10% FBS+P/S) 24 h prior to being transfected with pEGFP-N1 or pHSF1-GFPN3 (Wang et al., “Regulation of Molecular Chaperone Gene Transcription Involves the Serine Phosphorylation, 14-3-3 Epsilon Binding, and Cytoplasmic Sequestration of Heat Shock Factor 1,” Mol. Cell. Biol. 23:6013-6026 (2003), which is hereby incorporated by reference in its entirety) (gift from Stuart Calderwood, Addgene #32538) plasmid and Fugene HD reagent at 3:1 ratio. It should be noted that in this study for HCT116 and S2 cells, GFP or GFP-fusion protein refers to protein containing enhanced (E)-GFP which has equivalent binding affinity to the GFP-aptamer (Shui et al., “RNA Aptamers that Functionally Interact with Green Fluorescent Protein and its Derivatives,” Nucleic Acids Res. 40:e39 (2012), which is hereby incorporated by reference in its entirety). Transfection efficiency was monitored after 20 h (˜90% efficient as judged by GFP fluorescence) and cells were then subjected to instantaneous HS (Mahat et al., “Use of Conditioned Media is Critical for Studies of Regulation in Response to Rapid Heat Shock,” Cell Stress Chaperones 22:155-162 (2017), which is hereby incorporated by reference in its entirety). Cells were scraped after 30 min HS and centrifuged at 500×g for 5 min at 4.0 and washed twice with ice-cold 1× PBS.
Drosophila S2 cells were grown in M3+BPYE media (with 10% FBS) at 25.C. S. pombe cells were cultivated using standard procedures (Moreno et al., “Molecular Genetic Analysis of Fission Yeast Schizosaccharomyces pombe,” Methods Enzymol. 194:795-823 (1991), which is hereby incorporated by reference in its entirety).
Human cellular lysate preparation and aptamer-based affinity purification. Transfected human HCT116 cells before or after HS were resuspended in 0.5 ml ice-cold cellular lysis buffer (1× PBS+0.2% NP40+1× EDTA-free Protease inhibitor cocktail). Cells were incubated on ice for 30 min followed by sonication with Bioruptor Diagenode at High setting (30 s ON/30 s OFF) for 5 min at 4.C. The lysate was centrifuged at 20 000× g for 10 min at 4.0 and the resulting supernatant was transferred to a new tube. The cleared lysate was diluted to a final buffer containing 1×PBS, 0.05% NP40, 5.25 mM MgCl2, 187.5 ng/μl yeast RNA, 187.5 ng/μl sheared salmon sperm DNA, 200 units SUPERase IN/ml.
RNA preparation and immobilization on beads. The polyadenylated (20 nt ‘A’) GFP or polyadenylated control (NELF)-aptamer was in vitro transcribed using T7 RNA polymerase and purified by phenol/chloroform and polyacrylamide gel extraction. DNA sequences of the GFP and control (NELF)-RNA aptamers are based on sequences used in (Tome et al., “Comprehensive Analysis of RNA-protein Interactions by High-throughput Sequencing-RNA Affinity Profiling,” Nat. Methods 11:683-688 (2014), which is hereby incorporated by reference in its entirety) and are as follows; GFP-aptamer:
200 pmol of polyadenylatedGFP or the control (NELF)-aptamer was annealed to equimolar desthiobiotin-oligodT-20 in 200 of 1× annealing buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl) by heating at 95.0 for 3 min and slow cooling to room temperature over >1 h. For each pull-down 1 mg of Dynabeads MyOne Streptavidin C1 (Thermo) magnetic beads were washed once with 1 ml and twice with 0.1 ml of Tween wash buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20) by placing on a magnetic separator for 2 min and removing the supernatant. To eliminate possible RNase activity, beads were washed once with 0.1 ml of 0.1M NaOH, 0.05M NaCl followed by two washes of 0.1 ml of 0.1 M NaCl with changing tubes in between washes. The beads were resuspended in 200 μl of 2× binding buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2M NaCl) supplemented with 4 units/ml SUPERase IN. The resulting bead slurry was mixed with the annealed RNA aptamer and incubated on a thermomixer for 1 h at 23.0 with shaking. Aptamer bound beads were washed twice with 0.4 ml of Bead wash buffer (1× PBS, 0.05% NP40, 5 mM MgCl2), supplemented with 4 units/ml SUPERase IN with changing tubes in between washes.
Binding to lysate, washing and elution. The GFP- or control-aptamer bound beads were resuspended in the diluted cellular lysate and incubated at 4.0 with rotation for 2 h. The beads were placed on a magnet and supernatant was removed. The beads were washed twice with 0.5 ml of Bead wash buffer and twice with 0.5 ml of 1× PBS, 5 mM MgCl2, with changing tubes in between washes. Beads were resuspended in 50 μl of fresh Elution buffer (5 mM Biotin, 50 mM ammonium phosphate, pH 7.5) and incubated in a Thermomixer at 37.0 with shaking for 1 h followed by collection of the eluate into a fresh tube.
Details of the immunoprecipitation method utilizing the GFP-antibody are provided below.
Affinity purification from other organisms. Drosophila S2 cells stably expressing GFP, wild-type Drosophila S2 cells and S. pombe cells expressing endogenous GFP-Rpb3 were cultured for lysate or nuclear extract preparation, and were subjected to aptamer-based purification or Tandem affinity purification (TAP)-tag-based purification. Further details of these methods are provided below.
Proteomics workflow. Mass spectrometry sample preparation. Eluate from human HCT116 cells was solubilized in 8 M urea in 100 mM ammonium bicarbonate (ABC). The sample was reduced in 10 mM dithiothreitol (DTT) at 37.0 for 1 h. Cysteines were blocked in 30 mM methyl methanethiosulfonate (MMTS) at room temperature for 1 h without light. The sample volume was adjusted to reduce the concentration of urea to below 1M using 100 mM ABC, and proteins were digested using 1 μg of Trypsin at 37.0 overnight. Salts, RNA and other contaminants were removed using mixed-mode cation exchange (MCX) columns (Oasis). Elution buffer spiked-in with biotin and analyzed before and after MCX cleanup shows that the biotin signal is drastically reduced after cleanup (
Mass Spectrometry. All mass spectrometry with samples prepared from human HCT116 cells was performed on a Q-Exactive HF-X (Thermo Fisher Scientific) mass spectrometer with a EasyLC 1200 HPLC and autosampler (Thermo Fisher Scientific). The dried pull-downs were solubilized in 30 μl of loading buffer (0.1% trifluoroacetic acid and 2% acetonitrile in water), and 3 μl was injected via the autosampler onto a 150-μm Kasil fritted trap packed with Reprosil-Pur C18-AQ (3-μm bead diameter) to a bed length of 2 cm at a flow rate of 2 μl/min. After loading and desalting using a total volume of 8 μl of loading buffer, the trap was brought on-line with a pulled fused-silica capillary tip (75 μm i.d.) packed to a length of 25 cm with the same Dr. Maisch beads. The column and trap were mounted to a heated microspray source (CorSolutions) at 50° C. Peptides were eluted off of the column using a gradient of 5-28% acetonitrile in 0.1% formic acid over 25 min, followed by 28-60% acetonitrile over 5 min at a flow rate of 300 nl/min.
The mass spectrometer was operated using electrospray ionization (2 kV) with the heated transfer tube at 250.0 using data dependent acquisition (DDA), whereby one orbitrap mass spectrum (m/z 400-1600) was acquired with up to 20 orbitrap MS/MS spectra. The resolution for MS in the orbitrap was 60 000 at m/z 200, and 15 000 for MS/MS. The automatic gain control targets for MS was 3e6, and 1e5 for MS/MS. The maximum fill times were 45 and 25 ms, respectively. The MS/MS spectra were acquired using quadrupole isolation with an isolation width of 1.6 m/z and HCD collision energy (NCE) of 28%. The precursor ion threshold intensity was set to 2e6 in order to trigger an MS/MS acquisition. Furthermore, MS/MS acquisitions were allowed for precursor charge states of 2-5. Dynamic exclusion (including all isotope peaks) was set for 10 s.
Details of the mass spectrometry method associated with NELF-aptamer pull-down from Drosophila S2 cells are provided below.
Data Analysis. Raw spectral files were converted to mascot generic format using MSGUI, then searched against a database containing human proteins from UniProt with the addition of the protein sequence for GFP using Mascot. The search parameters allowed for fixed cysteine methylthiolation and variable methionine oxidation modifications, with a 10 ppm peptide mass tolerance, 0.5 Da fragment mass tolerance, and one missed tryptic cleavage. Subsequent searches allowed for variable lysine acetylation and serine/threonine phosphorylation, respectively, each with fixed cysteine methylthiolation and variable methionine oxidation allowing for 20 ppm peptide mass tolerance and 0.5 Da fragment mass tolerance and a maximum of three missed tryptic cleavages. Searches were also submitted with the above parameters and a 0.02 Da fragment mass tolerance, which resulted in no substantial changes to the results. Initial analyses of abundance and enrichment were conducted using Scaffold (Searle, “Scaffold: a Bioinformatic Tool for Validating MS/MS-based Proteomic Studies,” Proteomics 10: 1265-1269 (2010), which is hereby incorporated by reference in its entirety). Prediction and scoring of HSF1 interacting partners was done using Significance analysis of interactome (SAINT), and data was presented using the SAINT score and fold change A values (Choi et al., “SAINT: Probabilistic Scoring of Affinity Purification-mass Spectrometry Data,” Nat. Methods 8:70-73 (2011), which is hereby incorporated by reference in its entirety). Rather than performing a traditional fold change calculation, SAINT takes into account representation of each protein among biological replicates. As a control dataset to train the algorithm, the proteins detected in pull-downs using the GFP aptamer from GFP cells were used in addition to pull-downs using the NELF-aptamer from HSF1-GFP cells. This 2-fold control strategy provided proteins that bind non-specifically to RNA and to free GFP. The resulting output was an enrichment calculation (fold change A, the most conservative option) and probability score for interaction with HSF1 (SAINT score). Prediction and assignment of posttranslational modifications was done using Scaffold PTM (Vincent-Maloney et al., “Probabilistically Assigning Sites of Protein Modification with Scaffold PTM,” J. Biomol. Tech. 22:S36-S37 (2011), which is hereby incorporated by reference in its entirety). Site assignments were confirmed using MS1 quantification in Skyline (Pino et al., “The Skyline Ecosystem: Informatics for Quantitative Mass Spectrometry Proteomics,” Mass Spectrom. Rev. 39:229-244 (2017), which is hereby incorporated by reference in its entirety). All mass spectrometry proteome data were deposited to the ProteomeXchange Consortium (Deutsch et al., “The ProteomeXchange Consortium in 2017: Supporting the Cultural Change in Proteomics Public Data Deposition,” Nucleic Acids Res. 45:D1100-D1106 (2017), which is hereby incorporated by reference in its entirety) via the PRIDE repository (Perez-Riverol et al., “The PRIDE Database and Related Tools and Resources in 2019: Improving Support for Quantification Data,” Nucleic Acids Res. 47:D442-D450 (2019), which is hereby incorporated by reference in its entirety) with the dataset identifier PXDO15620.
Gene ontology (GO) analysis. The Protein Analysis Through Evolutionary Relationships (PANTHER) classification system (Mi et al., “Protocol Update for Large-scale Genome and Gene Function Analysis with the PANTHER Classification System (v.14.0),” Nat. Protoc. 14:703-721 (2019), which is hereby incorporated by reference in its entirety) was used to determine the GO classification of enriched proteins. UniProt IDs from proteins statistically enriched (Fishers exact test P<0.05) in HSF1-GFP cells compared to GFP cells with the GFP- or NELF-aptamer were used as query for the PANTHER 15.0 Gene List Analysis tool. Functional classification using the Homo sapiens reference database was performed using this tool. The percent of genes from the query matching to a specific function against the total number of queried genes with function matches was plotted using gg-plot2 (Wickham, H. In: Ggplot2: elegant graphics for data analysis. second edn. Springer, Switzerland (2016), which is hereby incorporated by reference in its entirety).
Immunoprecipitation of HSF1-GFP with the GFP antibody. HCT116 cells were transfected with HSF1-GFP plasmid and harvested after 20 hr of transfection. Cells were incubated in ice-cold cellular lysis buffer (1× PBS+0.2% NP40+1× EDTA-free Protease inhibitor cocktail) on ice for 30 min followed by sonication with Bioruptor Diagenode at High setting (30 s ON/30 s OFF) for 5 min at 4° C. The lysate was centrifuged at 20,000×g for 10 min at 4° C. and the resulting supernatant was transferred to a new tube. The cleared lysate was diluted to a final buffer containing 1× PBS, 0.05% NP40, 5.25 mM MgCl2. The GFP-antibody (Abcam, ab290) was immobilized on Protein-A Dynabeads for 4 h at 4° C. Beads were washed in antibody wash buffer (1× PBS, 5 mg/ml BSA, 0.05% Tween-20) and finally resuspended in the diluted cellular lysate with rotation at 4° C. for 2 h. The beads were washed twice in 0.5 ml bead wash buffer (1× PBS, 0.05% NP40, 5 mM MgCl2), with changing tubes in between washes. The beads were washed twice with 0.5 ml of 1× PBS, 5 mM MgCl2, with changing tubes in between washes. Beads were resuspended in 55 μL of 10 mM Tris-HCl, pH 8.5 and incubated in at 98° C. for 5 min followed by collection of the eluate into a fresh tube.
Aptamer-based GFP purification from Drosophila S2 cells. S2 cells stably expressing GFP were grown in M3+BPYE media (with 10% FBS) at 25° C. Around 100 million cells were collected by centrifugation at 1000×g for 5 min at 4° C. Cells were washed twice in 1× PBS and resuspended in 0.5 ml ice-cold cellular lysis buffer (1× PBS, 0.2% NP40, supplemented with 1× Protease inhibitor cocktail before use) and incubated on ice for 30 min followed by sonication with Bioruptor Diagenode at H setting (30 s ON/60 s OFF) for 15 min at 4° C. The lysate was centrifuged at 20,000×g for 10 min at 4° C. and the resulting supernatant was transferred to a new tube and diluted to a final buffer containing 1× PBS, 0.05% NP40, 5.25 mM MgCl2, 250 ng/μL yeast RNA, 250 ng/μL sheared salmon sperm DNA (sssDNA), 300 units/ml SUPERase IN.
For crosslinking experiments, 80 million cells were centrifuged at 1000× g for 10 min at 4° C. to remove the media and the pellet was resuspended in 1× PBS. Crosslinking was done by adding 1% Paraformaldehyde and incubated at room temperature for 10 min with occasional mixing. Glycine was added to a final concentration of 147 mM and incubated at room temperature for 5 min with occasional mixing. The crosslinked cells were centrifuged at 1000×g for 5 min at 4° C. and the supernatant was discarded. The pellet was washed with 1× PBS, flash frozen in liquid nitrogen and stored at −80° C. To prepare lysate from the crosslinked cells, the frozen cells were thawed on ice and resuspended in 0.5 ml of ice cold cellular lysis buffer. The cells were incubated on ice for 20 min and centrifuged at 4500×g for 5 min. The pellet was washed once more with 0.5 ml of ice cold cellular lysis buffer. The pellet was resuspended in 0.2 ml of 0.5% SDS and incubated at 37° C. for 15 min. The volume was brought up to 0.5 ml with 1× PBS, 1× Protease inhibitor cocktail and sonicated with Bioruptor Diagenode at H setting (20 s ON/60 s OFF) for 30 min at 4° C. The lysate was centrifuged at 20,000×g for 10 min at 4° C. The resulting supernatant was transferred to a new tube and diluted to a final buffer containing 1× PBS, 250 ng/μL yeast RNA, 250 sssDNA, 0.025% SDS, 0.05% Triton X-100, 5.25 mM MgCl2, 300 units/ml SUPERase IN. The aptamer RNA bead preparation, binding, washing and elution were done similar to the procedure used for human HCT116 cells, except in case of crosslinked cells, the elution was done with RNase A/T1 cocktail treatment.
Drosophila nuclear extract preparation and aptamer-based affinity purification. Nuclear extract was prepared from Drosophila S2 cells following the protocol as described in (Brunner et al., “A High Quality Catalog of the Drosophila melanogaster Proteome,” Nat Biotechnol 25:576-583 (2007), which is hereby incorporated by reference in its entirety). Briefly, cells were grown to confluency in T150 flasks, collected into falcon tubes and were washed 4 times with 1× PBS by centrifuging at 1000× g at 4° C. for 10 min. Cells were lysed with 5 pellet volumes of ice-cold hypotonic lysis buffer (10 mM HEPES pH 7.6, 1.5 mM MgCl2, 10 mM KCl, supplemented with 1× Protease inhibitor cocktail and 0.5 mM DTT before use) and incubated on ice for 10 min. The cells were dounced on ice for 20 times with a dounce homogenizer and tight pestle. The lysate was centrifuged at 1000×g at 4° C. for 7 min. Supernatant was discarded and the pellet was washed again with ice cold hypotonic lysis buffer. Finally, the pellet was resuspended in 0.5 packed volumes of ice-cold high salt extraction buffer (20 mM HEPES pH 7.6, 25% glycerol, 1.5 mM MgCl2, 420 mM NaCl, supplemented with 1× Protease inhibitor cocktail and 0.5 mM DTT before use) and the lysate was stirred for 30 min at 4° C. on an ice bath. The lysate was centrifuged at 20,000× g for 20 min at 4° C. and the supernatant containing the nuclear proteins was transferred into a fresh tube, flash frozen in liquid nitrogen and stored at −80° C. Before aptamer precipitation the nuclear extract was diluted to a final buffer containing 25 mM HEPES pH 7.6, 6.25% glycerol, 5 mM MgCl2, 105 mM NaCl, 25 mM KCl, 250 ng/μL yeast RNA, 250 ng/μL sssDNA and 50 μM Dextran sulfate.
RNA preparation and immobilization on beads: The in vitro transcribed, polyadenylated NELF- or polyadenylated control (GFP)-aptamer was annealed to desthiobiotin oligodT-20 and immobilized on the beads following the steps as described previously for human HCT116 cells by replacing the bead wash buffer with NE bead wash buffer (25 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 100 mM NaCl, 25 mM KCl, 0.05% NP40).
Binding to lysate, washing & elution: The NELF- or control-aptamer bound beads were resuspended in the diluted nuclear extract and incubated at 4° C. with rotation for 2 hr. The beads were placed on a magnet and supernatant was removed. The beads were washed twice with the NE bead wash buffer and twice with non-NP40 containing NE bead wash buffer with changing tubes in between washes. Beads were resuspended in 50 μL of fresh NE elution buffer (4 mM Biotin, 20 mM Tris-HCl, pH 7.5, 50 mM NaCl) and incubated in a Thermomixer at 37° C. with shaking for 1 hr followed by collection of the eluate into a fresh tube.
Aptamer-based GFP-Rpb3 purification from S. pombe cells. S. pombe strains used in this study has the genotype ade6-m216, ura4-D18, leu1, gfp-rpb3 (Source: National Bio Resource Project, Japan). Lysate preparation from fission yeast was performed as described previously (Kajitani et al., “Ser7 of RNAPIICTD Facilitates Heterochromatin Formation by Linking ncRNA to RNAi,” Proc Natl Acad Sci USA 114: E11208-E11217 (2017), which is hereby incorporated by reference in its entirety) with several modifications. Cells were cultured in 100 ml of YES to 1×107 cells/ml and harvested by centrifugation. Cell pellets were suspended in Lysis Buffer (1× PBS, 0.2% NP40, 1 mM PMSF, 1/50 volume of cOmpleteTM, Mini, EDTA-free Protease Inhibitor Cocktail [Roche], and 1/100 volume of PhosSTOPTM phosphatase inhibitor tablets [Roche]). Cell lysates were prepared by gentle vortexing with zirconia beads (Biospec products) in a FastPrep-24TM (Molecular Bio Products) at room temperature. Crude lysates were cleared by two-step centrifugation at 1100×g for 1 min at 4° C. and at 15,000×g for 10 min at 4° C. Lysates were diluted to a final buffer containing 1× PBS, 187 ng/μL yeast RNA, 187 ng/μL sssDNA, 200 units/ml SUPERase IN, 0.05% NP40, 5 mM MgCl2. The aptamer RNA bead preparation, binding, washing and elution were done similar to the procedure used for human HCT116 cells. Western blot was performed with GFP antibody (Abcam ab290, 1:2000 dilution) and secondary Licor antibody (anti-rabbit IRDye 800CW).
Tandem affinity purification (TAP)-tag-based purification of Drosophila HSF (dHSF) from Drosophila S2 cells. Drosophila S2 cells stably transfected with plasmids encoding either N- or C terminal TAP-tagged dHSF in pMK33-NTAP or pMK33-CTAP vectors (Veraksa et al., “Analyzing protein complexes in Drosophila with tandem affinity purification-mass spectrometry,” Dev Dyn 232:827-834 (2005), which is hereby incorporated by reference in its entirety) were selected and cultured in M3+BPYE media supplemented with 10% FBS, Penn/Strep, and 300 μg/ml Hygromycin. Protein expression was induced with 0.5 mM CuSO4 for 6 h at 24° C. Cells were collected by centrifugation at 3000 rpm, 10 min in 4° C., washed with ice-cold 1× PBS, and lysed with TAP Lysis Buffer (1×108 cells/ml: 10% Glycerol, 50 mM HEPESKOH pH 7.6, 150 mM KCl, 2 mM EDTA, 0.3% NP-40, freshly supplemented with 2 mM DTT, 1 mM PMSF, and 1× complete Protease inhibitor cocktail, 10 mM NaF). Cell lysate was incubated on ice for 15 min, passed through 21 gauge needle 10 times, and cleared by centrifugation at 20800×g 15 min at 4° C. Cleared lysate was passed through IgG sepharose column (150 μL resin) twice on ice and column was washed three times with TEV Cleavage Buffer (10 mM HEPES-KOH pH 7.6, 150 mM NaCl, 0.1% NP-40, 0.5 mM EDTA freshly supplemented with 1 mM DTT). Proteins were eluted by TEV protease cleavage (330 μL total volume with 1× TEV Cleavage Buffer with 4.25 μg TEV Protease) at RT for 1 h. The eluate was diluted 4 fold with Calmodulin Binding Buffer (10 mM HEPES-KOH pH 7.6, 150 mM NaCl, 0.1% NP-40, 1 mM Imidazole, 1 mM MgOAc, 2 mM CaCl2) freshly supplemented with 10 mM 2-Mercaptoethanol), passed twice through 50 μL of pre-washed Ni-NTA superflow resin to remove His-tagged TEV protease, and flowthrough is supplemented with 4 mM CaCl2) and passed twice over 50 μL of pre-washed Calmodulin resin on ice. Resin was then washed four times with Calmodulin Binding Buffer, and bound proteins were eluted twice with 150 μL Calmodulin Binding Buffer containing 25 mM EGTA. Proteins in the combined eluate were visualized by SDSPAGE/Silver Staining and individual bands were subjected to Mass Spectrometric analysis by Cornell Proteomics Core Facility.
Liquid chromatography-tandem mass spectrometry analysis of NELF-aptamer pull-downs from Drosophila S2 cells. Dried tryptic peptides from pulldowns using the NELF-aptamer from S2 cells were solubilized with 2% acetonitrile (ACN)/0.5% formic acid (FA) for nano LC-MS/MS analysis, which was carried out using an Orbitrap FusionTM TribridTM (Thermo-Fisher Scientific, San Jose, Calif.) mass spectrometer with a nanospray Flex Ion Source, and coupled with a Dionex UltiMate3000RSLCnano system (Thermo, Sunnyvale, Calif.). 5 μL of sample was injected onto a PepMap C-18 RP nano trap column (100 μm×20 mm, 5 μm, Dionex) with nanoViper Fittings via 20 μL/min trapping flow rate for on-line desalting, then separated on a PepMap C-18 RP nano column (75 μm×25 cm, 2 μm) at 35° C., and then eluted by a 60 min gradient of 5-35% ACN in 0.1% FA at 300 nL/min, followed by a 7 min ramp to 90% ACN/0.1% FA, and an 8 min hold at 90% ACN/0.1% FA. The column was re-equilibrated with 5% ACN/0.1% FA for 21 min before each subsequent injection. The Orbitrap Fusion was operated in positive ion mode with spray voltage set at 1.7 kV and source temperature 275° C. External calibration for Fourier transform (FT), ion trap and quadrupole mass analyzers was performed. In data dependent acquisition (DDA) analysis, the instrument was operated using the FT mass analyzer in MS scan to select precursor ions followed by 3 s “Top Speed” data-dependent CID ion trap MS/MS scans at 1.6 m/z quadrupole isolation for precursor peptides with multiply charged ions (+2-7) above a threshold ion count of 5,000 and using normalized collision energy of 30%. MS survey scans were at a resolving power of 120,000 (fwhm at m/z 200) for the mass range of m/z 375-1575. Dynamic exclusion parameters were set at repeat count 1 with a 40 s repeat duration and ±10 ppm exclusion mass width. The activation time was 10 ms for CID analysis. All data were acquired under Xcalibur 3.0 operation software (Thermo-Fisher Scientific).
The GFP-aptamer (Shui et al., “RNA Aptamers that Functionally Interact with Green Fluorescent Protein and its Derivatives,” Nucleic Acids Res. 40:e39 (2012), which is hereby incorporated by reference in its entirety) with polyA-tail is immobilized on streptavidin magnetic Dynabeads via hybridization with desthiobiotin-oligodT and is incubated with a cellular lysate expressing GFP-tagged POI. After gently washing the beads, the aptamer-bound proteins can be specifically eluted with excess biotin, which has much higher affinity for streptavidin and readily competes off desthiobiotin. Eluted proteins are then analyzed by MS, leaving non-specifically bound proteins on beads (
The AptA-MS method was tested by purifying HSF1 and its interacting partners from HCT116 cells transiently expressing HSF1 fused to GFP. HSF1 is a major regulator of stress-induced transcription that binds to hundreds of Heat Shock Elements (HSEs) genome-wide upon activation (Vihervaara et al., “Transcriptional Response to Stress is Pre-wired by Promoter and Enhancer Architecture,” Nat. Commun. 8:255 (2017), which is hereby incorporated by reference in its entirety). In non-heat shock (NETS) conditions, endogenous HSF1 is predominantly in an inactive monomeric state but converts to an active trimeric DNA-binding state upon HS (Vihervaara et al., “Molecular Mechanisms Driving Transcriptional Stress Responses,” Nat. Rev. Genet. 19:385-397 (2018), which is hereby incorporated by reference in its entirety). HSF1 is also regulated by chaperones and PTMs, and the identification of these binding partners and PTMs is critical to the understanding of HSF1's function (Gomez-Pastor et al., “Regulation of Heat Shock Transcription Factors and Their Roles in Physiology and Disease,” Nat. Rev. Mol. Cell Biol. 19:4-19 (2018); Vihervaara et al., “HSF1 at a Glance,” J. Cell Sci. 127:261-266 (2014), which are hereby incorporated by reference in their entirety). HCT116 cells were transfected with plasmid vectors expressing GFP or HSF1-GFP under the same promoter, and subjected to a 30 min heat shock at 42.0 (HS) or kept at 37.0 (NETS) (
To identify the interactors of the target proteins, the eluates were processed for MS and peptide spectra were analyzed using a bioinformatics pipeline that quantifies protein enrichment and identifies interacting partners as well as PTMs (
28 proteins were identified that are predicted to interact with HSF1 with high confidence based on SAINT score (Choi et al., “SAINT: Probabilistic Scoring of Affinity Purification-mass Spectrometry Data,” Nat. Methods 8:70-73 (2011), which is hereby incorporated by reference in its entirety), many of which are specific to the HS condition (
HSF1 has been previously shown to interact with translation elongation factor eEF1A1 based on an immunoprecipitation assay, which is implicated to have a broad regulatory function in the HS response (Vera et al., “The Translation Elongation Factor eEF1A1 Couples Transcription to Translation During Heat Shock Response,” Elife, 3:e03164 (2014)). Interestingly, AptA-MS identified not only eEF1A1, but also eEF1A2 (SAINT scores of 0.69 and 0.66, respectively) as HSF1-interactors. In addition, novel, high-confidence HSF1 interactions with translation elongation factor HBS1 and thymidylate kinase (a nucleotide biosynthesis enzyme) (SAINT scores of 0.92 and 0.98, respectively) have been identified during HS (
HSF1 is well-known to possess multiple PTMs in normal and HS conditions that include acetylation, phosphorylation and sumoylation of specific residues (Xu et al., “Post-translational Modification of Human Heat Shock Factors and Their Functions: a Recent Update by Proteomic Approach,” J. Proteome Res. 11:2625-2634 (2012), which is hereby incorporated by reference in its entirety). The pulldown strategy allowed identification of the acetylation and phosphorylation of HSF1 and other co-precipitants without any specific enrichment for these modifications (
sapiens GN = HSF1 PE = 1 SV = 1 (+3)
sapiens GN = GOLGA4 PE = 1 SV = 1
sapiens GN = RBBP6 PE = 1 SV = 1
sapiens GN = ABL2 PE = 1 SV = 1
sapiens GN = CENPE PE = 1 SV = 2
sapiens GN = ELAVL4 PE = 1 SV = 2 (+1)
sapiens GN = HIST1H2BA PE = 1 SV = 3 (+1)
sapiens GN = KNTC1 PE = 1 SV = 1
sapiens GN = RPL22L1 PE = 1 SV = 2
sapiens GN = BRDT PE = 1 SV = 4
sapiens GN = RANBP2 PE = 1 SV = 2
sapiens GN = ILF3 PE = 1 SV = 3
sapiens GN = ATRX PE = 1 SV = 5
sapiens GN = UBA52 PE = 1 SV = 2
sapiens GN = C1orf14 PE = 1 SV = 2
sapiens GN = C17orf85 PE = 1 SV = 2
sapiens GN = ALS2CR8 PE = 2 SV = 2
sapiens GN = DHX9 PE = 1 SV = 4
sapiens GN = CDC5L PE = 1 SV = 2
sapiens GN = CSPG4 PE = 1 SV = 2
sapiens GN = UBR5 PE = 1 SV = 2
sapiens GN = YWHAQ PE = 1 SV = 1 (+3)
sapiens GN = RPS17 PE = 1 SV = 2 (+1)
sapiens GN = RPL3 PE = 1 SV = 2 (+1)
sapiens GN = SLC25A4 PE = 1 SV = 4 (+2)
sapiens GN = EEF1A1 PE = 1 SV = 1 (+1)
sapiens GN = HIST1H2AB PE = 1 SV = 2 (+2)
sapiens GN = HIST1H2BK PE = 1 SV = 3 (+6)
sapiens GN = C1orf57 PE = 1 SV = 1
sapiens GN = RBM15 PE = 1 SV = 2
sapiens GN = ARHGAP11A PE = 1 SV = 2
sapiens GN = ARHGAP28 PE = 2 SV = 3
sapiens GN = SAFB PE = 1 SV = 4
sapiens GN = SRSF3 PE = 1 SV = 1
sapiens GN = CHEK1 PE = 1 SV = 1
sapiens GN = ANKRD28 PE = 1 SV = 4
sapiens GN = SNW1 PE = 1 SV = 1
sapiens GN = HSPA9 PE = 1 SV = 2
sapiens GN = UBXN4 PE = 1 SV = 2
sapiens GN = KIAA0195 PE = 1 SV = 1
sapiens GN = KIAA1370 PE = 2 SV = 2
sapiens GN = SLC3A2 PE = 1 SV = 3
sapiens GN = PFKP PE = 1 SV = 2
sapiens GN = PSMC3 PE = 1 SV = 3
sapiens GN = PSMC4 PE = 1 SV = 2
sapiens GN = PSMC6 PE = 1 SV = 1
sapiens GN = RPS27L PE = 1 SV = 3
sapiens GN = RPLP0 PE = 1 SV = 1
sapiens GN = RPLP0P6 PE = 5 SV = 1
sapiens GN = RPL26L1 PE = 1 SV = 1
sapiens GN = TRIP4 PE = 1 SV = 4
sapiens GN = ABT1 PE = 1 SV = 1
sapiens GN = APEH PE = 1 SV = 4
sapiens GN = APRT PE = 1 SV = 2
sapiens GN = AK2 PE = 1 SV = 2
sapiens GN = AK1 PE = 1 SV = 3
sapiens GN = ADRA2B PE = 2 SV = 2
sapiens GN = AP1G2 PE = 1 SV = 1
sapiens GN = NAT1 PE = 1 SV = 2
sapiens GN = ATPIF1 PE = 1 SV = 1
sapiens GN = BCL11B PE = 1 SV = 1
sapiens GN = BCAP31 PE = 1 SV = 3
sapiens GN = C2CD3 PE = 1 SV = 4
sapiens GN = CLCF1 PE = 1 SV = 1
sapiens GN = CSNK1G3 PE = 1 SV = 2
sapiens GN = CSNK2A1 PE = 1 SV = 1
sapiens GN = CCPG1 PE = 1 SV = 3
sapiens GN = CNBP PE = 1 SV = 1
sapiens GN = CS PE = 1 SV = 2
sapiens GN = COMMD9 PE = 1 SV = 2
sapiens GN = C1QBP PE = 1 SV = 1
sapiens GN = ATP7B PE = 1 SV = 4
sapiens GN = CFDP1 PE = 1 SV = 1
sapiens GN = UQCRB PE = 1 SV = 2
sapiens GN = MT-CO2 PE = 1 SV = 1
sapiens GN = DYNC1H1 PE = 1 SV = 5
sapiens GN = DYNC2H1 PE = 1 SV = 4
sapiens GN = CKAP2 PE = 1 SV = 1
sapiens GN = CKAP4 PE = 1 SV = 2
sapiens GN = DAPK2 PE = 1 SV = 1
sapiens GN = DUT PE = 1 SV = 3
sapiens GN = DCLRE1A PE = 1 SV = 3
sapiens GN = MSH3 PE = 1 SV = 3
sapiens GN = DDOST PE = 1 SV = 4
sapiens GN = CLK4 PE = 1 SV = 1
sapiens GN = DNAH1 PE = 1 SV = 3
sapiens GN = PIAS2 PE = 1 SV = 3
sapiens GN = RNF20 PE = 1 SV = 2
sapiens GN = HUWE1 PE = 1 SV = 3
sapiens GN = RAD18 PE = 1 SV = 2
sapiens GN = TRIM63 PE = 1 SV = 1
sapiens GN = TUFM PE = 1 SV = 2
sapiens GN = ERH PE = 1 SV = 1
sapiens GN = EIF4A1 PE = 1 SV = 1
sapiens GN = EIF4A2 PE = 1 SV = 2
sapiens GN = EIF4A3 PE = 1 SV = 4
sapiens GN = CAPZB PE = 1 SV = 4
sapiens GN = SSRP1 PE = 1 SV = 1
sapiens GN = FANCI PE = 1 SV = 4
sapiens GN = ALDOC PE = 1 SV = 2
sapiens GN = FH PE = 1 SV = 3
sapiens GN = QSER1 PE = 1 SV = 3
sapiens GN = GSR PE = 1 SV = 2
sapiens GN = GSTO1 PE = 1 SV = 2
sapiens GN = YWHAB PE = 1 SV = 3 (+2)
sapiens GN = RPS10 PE = 1 SV = 1 (+1)
sapiens GN = RPL10 PE = 1 SV = 4 (+1)
sapiens GN = ANP32C PE = 2 SV = 1 (+3)
sapiens GN = CSDA PE = 1 SV = 4 (+1)
sapiens GN = ST13 PE = 1 SV = 2 (+2)
sapiens GN = PPA1 PE = 1 SV = 2 (+1)
sapiens GN = MYL6B PE = 1 SV = 1 (+1)
sapiens GN = PGK1 PE = 1 SV = 3 (+1)
sapiens GN = PGAM2 PE = 1 SV = 3 (+1)
sapiens GN = RAB13 PE = 1 SV = 1 (+4)
sapiens GN = TPI1 PE = 1 SV = 2 (+1)
sapiens GN = TUBAL3 PE = 1 SV = 2 (+2)
sapiens GN = TUBA1B PE = 1 SV = 1 (+2)
sapiens GN = TUBA4A PE = 1 SV = 1 (+2)
sapiens GN = TUBB4 PE = 1 SV = 2 (+5)
sapiens GN = RAN PE = 1 SV = 3
sapiens GN = ERAL1 PE = 1 SV = 2
sapiens GN = GNB1 PE = 1 SV = 3
sapiens GN = HSPA4L PE = 1 SV = 3
sapiens GN = HSPA12B PE = 1 SV = 2
sapiens GN = HDGF PE = 1 SV = 1
sapiens GN = HMG20A PE = 1 SV = 1
sapiens GN = HMGB1 PE = 1 SV = 3
sapiens GN = HNMT PE = 1 SV = 1
sapiens GN = MYST2 PE = 1 SV = 1
sapiens GN = UNCX PE = 2 SV = 1
sapiens GN = HYOU1 PE = 1 SV = 1
sapiens GN = IP6K1 PE = 1 SV = 3
sapiens GN = ISYNA1 PE = 1 SV = 1
sapiens GN = ITPK1 PE = 1 SV = 2
sapiens GN = ILF2 PE = 1 SV = 2
sapiens GN = IL1F9 PE = 1 SV = 1
sapiens GN = KHDRBS1 PE = 1 SV = 1
sapiens GN = KHDRBS3 PE = 1 SV = 1
sapiens GN = BAT2 PE = 1 SV = 3
sapiens GN = LSG1 PE = 1 SV = 2
sapiens GN = SHOC2 PE = 1 SV = 2
sapiens GN = LASP1 PE = 1 SV = 2
sapiens GN = MPP3 PE = 1 SV = 2
sapiens GN = MPP6 PE = 1 SV = 2
sapiens GN = MDH1 PE = 1 SV = 4
sapiens GN = MAGEB10 PE = 2 SV = 4
sapiens GN = MAGED2 PE = 1 SV = 2
sapiens GN = PAQR8 PE = 2 SV = 1
sapiens GN = MFAP1 PE = 1 SV = 2
sapiens GN = MAP4 PE = 1 SV = 3
sapiens GN = BUB3 PE = 1 SV = 1
sapiens GN = PAICS PE = 1 SV = 3
sapiens GN = L1CAM PE = 1 SV = 2
sapiens GN = NEFM PE = 1 SV = 3
sapiens GN = NEUROD4 PE = 2 SV = 2
sapiens GN = GANAB PE = 1 SV = 3
sapiens GN = SLC1A5 PE = 1 SV = 2
sapiens GN = NBR1 PE = 1 SV = 3
sapiens GN = NAIF1 PE = 1 SV = 1
sapiens GN = NASP PE = 1 SV = 2
sapiens GN = NUDC PE = 1 SV = 1
sapiens GN = NUMA1 PE = 1 SV = 2
sapiens GN = GTPBP4 PE = 1 SV = 3
sapiens GN = GNL2 PE = 1 SV = 1
sapiens GN = PCID2 PE = 1 SV = 2
sapiens GN = PPIB PE = 1 SV = 2
sapiens GN = PRDX5 PE = 1 SV = 3
sapiens GN = PSAT1 PE = 1 SV = 2
sapiens GN = PARP1 PE = 1 SV = 4
sapiens GN = SF3B14 PE = 1 SV = 1
sapiens GN = PRPF6 PE = 1 SV = 1
sapiens GN = PRPF19 PE = 1 SV = 1
sapiens GN = RBM22 PE = 1 SV = 1
sapiens GN = SLU7 PE = 1 SV = 2
sapiens GN = BCAS2 PE = 1 SV = 1
sapiens GN = XAB2 PE = 1 SV = 2
sapiens GN = WTAP PE = 1 SV = 2
sapiens GN = TARBP1 PE = 1 SV = 1
sapiens GN = PDCD5 PE = 1 SV = 3
sapiens GN = PCNA PE = 1 SV = 1
sapiens GN = PA2G4 PE = 1 SV = 3
sapiens GN = PSMA1 PE = 1 SV = 1
sapiens GN = PSMA2 PE = 1 SV = 2
sapiens GN = PSMA3 PE = 1 SV = 2
sapiens GN = PSMA5 PE = 1 SV = 3
sapiens GN = PSMA6 PE = 1 SV = 1
sapiens GN = PSMA7 PE = 1 SV = 1
sapiens GN = PSMB1 PE = 1 SV = 2
sapiens GN = PSMB2 PE = 1 SV = 1
sapiens GN = PSMB5 PE = 1 SV = 3
sapiens GN = PSMB6 PE = 1 SV = 4
sapiens GN = PSMB7 PE = 1 SV = 1
sapiens GN = PDIA3 PE = 1 SV = 4
sapiens GN = PDIA4 PE = 1 SV = 2
sapiens GN = PDIA6 PE = 1 SV = 1
sapiens GN = MAGOHB PE = 1 SV = 1
sapiens GN = PRC1 PE = 1 SV = 2
sapiens GN = SEC31B PE = 1 SV = 1
sapiens GN = NPEPPS PE = 1 SV = 2
sapiens GN = AHCYL1 PE = 1 SV = 2
sapiens GN = NACAP1 PE = 5 SV = 1
sapiens GN = OR56B2P PE = 5 SV = 1
sapiens GN = DHX15 PE = 1 SV = 2
sapiens GN = FTSJ3 PE = 1 SV = 2
sapiens PE = 2 SV = 1
sapiens GN = GDI1 PE = 1 SV = 2
sapiens GN = GDI2 PE = 1 SV = 2
sapiens GN = RANGAP1 PE = 1 SV = 1
sapiens GN = RGS14 PE = 1 SV = 4
sapiens GN = RFC4 PE = 1 SV = 2
sapiens GN = ARHGDIA PE = 1 SV = 3
sapiens GN = BOP1 PE = 1 SV = 2
sapiens GN = RALYL PE = 1 SV = 2
sapiens GN = ESD PE = 1 SV = 2
sapiens GN = SLTM PE = 1 SV = 2
sapiens GN = SRSF2 PE = 1 SV = 4
sapiens GN = SRSF5 PE = 1 SV = 1
sapiens GN = SRSF7 PE = 1 SV = 1
sapiens GN = SRSF8 PE = 1 SV = 1
sapiens GN = PRKD3 PE = 1 SV = 1
sapiens GN = UHMK1 PE = 1 SV = 2
sapiens GN = PPP2R1A PE = 1 SV = 4
sapiens GN = PPP2CB PE = 1 SV = 1
sapiens GN = SARS PE = 1 SV = 3
sapiens GN = SIRPB2 PE = 2 SV = 1
sapiens GN = SNRPE PE = 1 SV = 1
sapiens GN = SNRPG PE = 1 SV = 1
sapiens GN = SUMO3 PE = 1 SV = 2
sapiens GN = SUMO4 PE = 1 SV = 2
sapiens GN = SLC12A3 PE = 1 SV = 3
sapiens GN = UTP3 PE = 1 SV = 1
sapiens GN = SPTA1 PE = 1 SV = 5
sapiens GN = STIP1 PE = 1 SV = 1
sapiens GN = PPAN PE = 1 SV = 1
sapiens GN = SMN1 PE = 1 SV = 1
sapiens GN = TCP1 PE = 1 SV = 1
sapiens GN = CCT2 PE = 1 SV = 4
sapiens GN = CCT4 PE = 1 SV = 4
sapiens GN = CCT5 PE = 1 SV = 1
sapiens GN = CCT7 PE = 1 SV = 2
sapiens GN = CCT3 PE = 1 SV = 4
sapiens GN = CCT8 PE = 1 SV = 4
sapiens GN = CCT6A PE = 1 SV = 3
sapiens GN = CCT6B PE = 1 SV = 4
sapiens GN = ERG PE = 1 SV = 2
sapiens GN = TFRC PE = 1 SV = 2
sapiens GN = TRA2B PE = 1 SV = 1
sapiens GN = GPNMB PE = 1 SV = 2
sapiens GN = TBCA PE = 1 SV = 3
sapiens GN = TDRD3 PE = 1 SV = 1
sapiens GN = UBAP2L PE = 1 SV = 2
sapiens GN = UBXN2A PE = 2 SV = 1
sapiens GN = UGT2B7 PE = 1 SV = 1
sapiens GN = UHRF1BP1L PE = 1 SV = 2
sapiens GN = C1orf103 PE = 1 SV = 1
sapiens GN = C1orf106 PE = 2 SV = 1
sapiens GN = C2orf42 PE = 2 SV = 1
sapiens GN = C8orf42 PE = 2 SV = 2
sapiens GN = C10orf71 PE = 2 SV = 2
sapiens GN = C12orf43 PE = 1 SV = 2
sapiens GN = C14orf43 PE = 1 SV = 2
sapiens GN = C14orf166B PE = 2 SV = 2
sapiens GN = C17orf47 PE = 1 SV = 3
sapiens GN = C19orf34 PE = 2 SV = 2
sapiens GN = C20orf194 PE = 2 SV = 1
sapiens GN = KIAA1683 PE = 1 SV = 1
sapiens GN = WASH2P PE = 2 SV = 2
sapiens GN = WDR1 PE = 1 SV = 4
sapiens GN = WDR33 PE = 1 SV = 2
sapiens GN = WDR43 PE = 1 SV = 3
sapiens GN = WDR46 PE = 1 SV = 3
sapiens GN = WDR74 PE = 1 SV = 1
sapiens GN = WDR81 PE = 2 SV = 1
sapiens GN = WDR87 PE = 2 SV = 3
sapiens GN = YTHDC1 PE = 1 SV = 3
MS1 quantification in Skyline (Pino et al., “The Skyline Ecosystem: Informatics for Quantitative Mass Spectrometry Proteomics,” Mass Spectrom. Rev. 39:229-244 (2017), which is hereby incorporated by reference in its entirety) was used to validate the quality of the modified peptides (
32 proteins were identified in NHS cells expressing HSF1-GFP that are enriched compared to GFP expressing cells upon pull-down with the GFP-aptamer based on a Fisher's exact test using a P value cutoff of <0.05. In the same pulldowns from the HS cells, 42 enriched proteins were identified. Earlier interaction studies have not identified histones as co-precipitants in HSF1 immunoprecipitations or affinity purifications, potentially due to large variation in protein abundances in immunoprecipitated samples. Two histone proteins were found to be enriched in NHS cells, whereas six histone proteins were enriched in HS cells (Table 5). This illustrates that the chromatin landscape changes in response to HS, and that these changes are associated with HSF1-containing complexes. This fact is further demonstrated by the increase in proteins with ‘binding’ activity in HSF1 pull-downs from HS cells, as shown by GO analysis (
AptA-MS recapitulated published observations that HSF1 engages with chaperone proteins such as HSP70. It is found that AptA-MS pull-downs contain a large proportion of chaperone proteins in NHS and HS conditions (
Cytoskeletal reorganization and increased levels of transcription of cytoskeletal proteins have been observed after HS in some cell types, but their role and possible interplay with active HSF1 have not been documented (Walter et al., “Heat Shock Causes the Collapse of the Intermediate Filament Cytoskeleton in Drosophila Embryos,” Dev. Genet. 11:270-279 (1990), which is hereby incorporated by reference in its entirety). Upon HS an increase in HSF1-associated proteins with ‘structural molecule activity’ (
The rigorously controlled study identified proteins that bind to free GFP and to a control (NELF)-aptamer selected for Drosophila NELF-E (dNELF-E) (Pagano et al., “Defining NELF-E RNA Binding in HIV-1 and Promoter-proximal Pause Regions,” PLoS Genet. 10:e1004090 (2014); Tome et al., “Comprehensive Analysis of RNA-protein Interactions by High-throughput Sequencing-RNA Affinity Profiling,” Nat. Methods 11:683-688 (2014), which are hereby incorporated by reference in their entirety) with no predicted binding specificity in humans. A Fisher's exact test was used to statistically compare proteins enriched by the GFP-aptamer in cells expressing free GFP compared to cells expressing HSF1-GFP (Table 3).
sapiens GN = NOP2 PE = 1 SV = 2
sapiens GN = NONO PE = 1 SV = 4
sapiens GN = HNRNPA2B1 PE = 1 SV = 2
sapiens GN = RRS1 PE = 1 SV = 2
sapiens GN = RRP1 PE = 1 SV = 1
sapiens GN = DDX27 PE = 1 SV = 2
sapiens GN = HNRNPC PE = 1 SV = 4
sapiens GN = DBT PE = 1 SV = 3
sapiens GN = SNRNP200 PE = 1 SV = 2
sapiens GN = NOP2 PE = 1 SV = 2
sapiens GN = NONO PE = 1 SV = 4
sapiens GN = G3BP2 PE = 1 SV = 2
sapiens GN = G3BP1 PE = 1 SV = 1
sapiens GN = NAA38 PE = 1 SV = 3
sapiens GN = DDX23 PE = 1 SV = 3
sapiens GN = ZC3H13 PE = 1 SV = 1
sapiens GN = RRS1 PE = 1 SV = 2
sapiens GN = RRP1B PE = 1 SV = 3
sapiens GN = DDX47 PE = 1 SV = 1
Similarly, proteins were identified that are statistically enriched in pull-downs from HSF1-GFP expressing cells using the control-aptamer, compared to the GFP-aptamer (Table 3). These datasets are highly informative as they provide a list of common contaminants for an AptA-MS experiment. The first comparison generates a list of proteins that bind to GFP nonspecifically that can be used for future experiments using a GFP-tag. GO analysis of proteins enriched by the NELF-aptamer shows that the majority of these proteins function in binding, primarily to nucleic acids (
To test the broad applicability of AptA-MS, GFP was affinity enriched from Drosophila S2 cells expressing GFP with or without formaldehyde crosslinking (
Conventional affinity purification strategies rely upon the use of antibodies against a specific protein or epitope tag for successful enrichment of the bait protein and identification of its interacting partners (Bauer et al., “Affinity Purification-mass Spectrometry. Powerful Tools for the Characterization of Protein Complexes,” Eur. J. Biochem. 270:570-578 (2003), which is hereby incorporated by reference in its entirety). Nucleic acid-dependent affinity purification methods provide critical advantages over immunoprecipitations by limiting the amount of contaminating peptides that mask the detection of low-abundant interactors. Although relatively limited, aptamer-based purification strategies have been previously implemented for protein purification from biological sources (Perret et al., “Aptamer-based Affinity Chromatography for Protein Extraction and Purification,” In: Adv. Biochem. Eng. Biotechnol. Springer, Berlin, Heidelberg, pp. 1-47 (2019); Srisawat et al., “Streptavidin Aptamers: Affinity Tags for the Study of RNAs and Ribonucleoproteins,” RNA 7:632-641 (2001); Lonne et al., “Development of an Aptamer-based Affinity Purification Method for Vascular Endothelial Growth Factor,” Biotechnol. Rep. (Amst.) 8:16-23 (2015)). In addition, the Thrombin, IgE and ATP aptamers have been used for target isolation and detection by MS (Dick et al., “Aptamer-enhanced laser desorption/ionization for affinity mass spectrometry,” Anal. Chem. 76:3037-3041 (2004); Cole et al., “Affinity capture and detection of immunoglobulin E in human serum using an aptamer-modified surface in matrix-assisted laser desorption/ionization mass spectrometry,” Anal. Chem. 79:273-279 (2007); Ocsoy et al., “Aptamer-conjugated multifunctional nanoflowers as a platform for targeting, capture, and detection in laser desorption ionization mass spectrometry,” ACS Nano 7:417-427 (2013)). In one study, the EGFR and INSR aptamers were utilized to detect some of the interactors of the target proteins by western blot (Kim, et al., “Efficient Isolation and Elution of Cellular Proteins using Aptamer-mediated Protein Precipitation Assay,” Biochem. Biophys. Res. Commun. 448:114-119 (2014), which is hereby incorporated by reference in its entirety). However, aptamers have not been broadly used for exploring biological questions pertaining to protein-protein interactions utilizing MS. Furthermore, the previous aptamer-based analytical assays were restricted to aptamers against specific proteins of a particular species or small molecules, lacking the versatility and high sensitivity that is achievable by AptA-MS, as the latter takes advantage of GFP as an affinity tag. The GFP-aptamer was utilized to develop AptA-MS for the following reasons. First, GFP is a widely-used protein tag that has been applied in cellular imaging for decades. Therefore, an affinity purification approach targeting GFP would potentially serve as a common strategy for purifying hundreds of GFP-tagged proteins thereby minimizing technical variation and background. Second, GFP has no significant sequence similarity in human cells or commonly used model organisms making it less prone to non-specific interactions with other cellular components. Third, GFP has no known propensity for nucleic acids, which provides an explanation for the previous unsuccessful attempts of selecting an aptamer against it (Stanlis et al., “Single-strand DNA Aptamers as Probes for Protein Localization in Cells,” J. Histochem. Cytochem. 51:797-808 (2003), which is hereby incorporated by reference in its entirety). Therefore, the high-affinity aptamer against GFP should make it ‘less-sticky’ to other cellular proteins. Although, aptamers are considered to be ‘high-affinity’ reagents, the ligand binding affinities range from picomolar to micromolar dissociation constants (Kd) (Ilgu et al., “Aptamers in Analytics,” Analyst 141:1551-1568 (2016), which is hereby incorporated by reference in its entirety). Many of these aptamers cannot be used for affinity purification, as it is believed that the target-affinity requirement for such application should be at least in the low nanomolar range. In this aspect, the GFP-aptamer stands out due to its strong binding affinity (Kd=2.4-4.2 nM) (Tome et al., “Comprehensive Analysis of RNA-protein Interactions by High-throughput Sequencing-RNA Affinity Profiling,” Nat. Methods 11:683-688 (2014), which is hereby incorporated by reference in its entirety) and therefore, is suitable to serve the purpose. A good affinity-purification strategy requires reagents that are not only high-affinity, but also highly-specific and with broad utility. The GFP-tag and GFP-aptamer combination satisfies all these criteria and serves as a tool to study protein-protein interactions with high confidence, and is widely applicable in different species, tissues, and cell types.
To demonstrate the practical utility of the method, HSF1 was targeted, a critical regulator of HS response in mammals. HSF1 mediated activation of gene expression is associated with its binding to HSEs at pre-established transcriptional regulatory elements and release of paused PolII into productive elongation (Vihervaara et al., “Transcriptional Response to Stress is Pre-wired by Promoter and Enhancer Architecture,” Nat. Commun. 8:255 (2017); Mahat et al., “Mammalian Heat Shock Response and Mechanisms Underlying its Genome-wide Transcriptional Regulation,” Mol. Cell, 62:63-78 (2016); Ray et al., “Chromatin Conformation Remains Stable Upon Extensive Transcriptional Changes Driven by Heat Shock,” Proc. Natl Acad. Sci. U.S.A., 116:19431-19439 (2019), which are hereby incorporated by reference in their entirety). In normal conditions, HSF1 is constitutively expressed and remains inactive but upon stress it is converted to a transcriptionally active state, coordinated by PTMs and interactions with other proteins (Akerfelt et al., “Heat Shock Factors: Integrators of Cell Stress, Development and Lifespan,” Nat. Rev. Mol. Cell Biol. 11:545-555 (2010), which is hereby incorporated by reference in its entirety). Epitope-tagged HSF1 has been previously expressed in human cells to identify its PTMs and interacting partners upon immunoprecipitation followed by MS (Raychaudhuri et al., “Interplay of Acetyltransferase EP300 and the Proteasome System in Regulating Heat Shock Transcription Factor 1,” Cell 156:975-985 (2014); Fujimoto et al., “RPA assists HSF1 Access to Nucleosomal DNA by Recruiting Histone Chaperone FACT,” Mol. Cell 48:182-194 (2012), which are hereby incorporated by reference in their entirety). With AptA-MS some of the strong interactors and PTMs have been verified that have previously been detected but also have identified a few novel interacting partners/co-precipitates, clearly demonstrating the potential of this technology.
Differential protein purification analysis shows a major response to HS that is coordinated by HSF1. Like previous studies, chaperone proteins are detected that are HS responsive and interact with HSF1 to induce transcriptional events (Raychaudhuri et al., “Interplay of Acetyltransferase EP300 and the Proteasome System in Regulating Heat Shock Transcription Factor 1,” Cell 156:975-985 (2014), which is hereby incorporated by reference in its entirety). The functional classes associated with this induced interaction are illustrated via GO analysis showing an increased proportion of chaperone proteins and proteins with ‘binding’ activity to be interacting with HSF1 after HS. Furthermore, nucleic acid-binding proteins are more enriched after HS, and these proteins shift from primarily RNA-binding to mostly DNA-binding proteins in HS cells. This may reflect HSF1's increased DNA binding activity upon HS.
Expression of cytoskeletal genes has been shown to be up-regulated early during HS (Mahat et al., “Mammalian Heat Shock Response and Mechanisms Underlying its Genome-wide Transcriptional Regulation,” Mol. Cell 62:63-78 (2016), which is hereby incorporated by reference in its entirety), and interestingly an increased enrichment of cytoskeletal proteins copurifying with HSF1 is observed in these conditions. In particular, high-confidence interactions are demonstrated between HSF1 and cytoskeletal proteins including tubulin and keratin only after HS. It is speculated that these interactions could be a component of a feedback regulation that keeps the HS response modulated.
The chromatin environment of cells is also dramatically changed by stress. Alteration of histone PTM levels have been shown to be associated with HSF1 occupancy on chromatin upon HS (Vihervaara et al., “Transcriptional Response to Stress is Pre-wired by Promoter and Enhancer Architecture,” Nat. Commun 8:255 (2017); Kusch et al., “Histone H3 Lysine 4 trimethylation Regulates Cotranscriptional H2A Variant Exchange by Tip60 Complexes to Maximize Gene Expression,” Proc. Natl Acad. Sci. U.S.A., 111:4850-4855 (2014), which are hereby incorporated by reference in their entirety). Histone methyltransferases particularly targeting histone H3 lysine 4 have been shown to contribute to the HS response (Raychaudhuri et al., “Interplay of Acetyltransferase EP300 and the Proteasome System in Regulating Heat Shock Transcription Factor 1,” Cell 156:975-985 (2014); Ardehali et al., “Drosophila Set1 is the Major Histone H3 Lysine 4 Trimethyltransferase with Role in Transcription,” EMBO J. 30:2817-2828 (2011), which are hereby incorporated by reference in their entirety). While these histone modifiers may not directly interact with HSF1, a HS-induced copurification of histone H3 with HSF1 is shown, indicating that HSF1 is binding near transcriptionally active H3-containing DNA. In addition, induced interactions are detected between HSF1 and histones H2B and H4. The method can likely pick up these low-frequency interactions by virtue of its low background signal, and shows that HSF1 is binding differentially to transcriptionally-active chromatin as previously reported (Vihervaara et al., “Transcriptional Response to Stress is Pre-wired by Promoter and Enhancer Architecture,” Nat. Commun 8:255 (2017), which is hereby incorporated by reference in its entirety).
HSF1 has been shown to undergo extensive PTMs during its regulation. HSF1 is ubiquitinated during recovery from HS and when overexpressed in cells, and we find an interaction between HSF1 and a ubiquitin-40S protein, likely reflecting this modification process. Multiple lysines of HSF1 were found to be acetylated even in the NHS condition (Raychaudhuri et al., “Interplay of Acetyltransferase EP300 and the Proteasome System in Regulating Heat Shock Transcription Factor 1,” Cell 156:975-985 (2014), which is hereby incorporated by reference in its entirety), while acetylation of specific lysine residues was shown to be critical to the HS response. Acetylation of K80 and K118, was shown to be crucial for the release of HSF1 trimers from the HSEs and inhibited chromatin binding of HSF1 (Raychaudhuri et al., “Interplay of Acetyltransferase EP300 and the Proteasome System in Regulating Heat Shock Transcription Factor 1,” Cell 156:975-985 (2014); Westerheide et al., “Stress-inducible Regulation of Heat Shock Factor 1 by the Deacetylase SIRT1,” Science 323:1063-1066 (2009), which are hereby incorporated by reference in their entirety). In addition, acetylation of K208 by EP300 modulates HSF1 function and protein turnover. Using AptA-MS, acetylation has been detected at each of these essential residues in both NHS and HS conditions in addition to K62 and K162, providing an opportunity for further investigation to elucidate their roles in HSF1 regulation.
The HS response varies by tissue and cell type (Guisbert et al., “Identification of a Tissue-selective Heat Shock Response Regulatory Network,” PLoS Genet. 9:e1003466 (2013); van Oosten-Hawle, et al., “Organismal Proteostasis: Role of Cell-nonautonomous Regulation and Transcellular Chaperone Signaling,” Genes Dev. 28:1533-1543 (2014), which are hereby incorporated by reference in their entirety). Not only does AptA-MS provide a robust method to investigate MCs and their interactions, but it also gives a snapshot of the HSF1-mediated response to HS in HCT116 cells. These data complement previous work done in other cell lines and also give a unique insight into a cell-specific process that requires interaction of translation elongation factors and cytoskeletal proteins with HSF1 during HS response.
AptA-MS provides a detailed view of HSF1 interactions and modifications before and after HS. In addition, control pull-downs also reveal essential information. Pull-downs using GFP only cells with GFP-aptamer or HSF1-GFP cells with a structured control RNA aptamer (selected against dNELF-E) with no predicted binding partners in HCT116 cells provide a proteomic profile for nonspecific interactions between RNA and proteins. The proteins identified from GFP only cells subjected to GFP-aptamer pull-down are not only informative about the nonspecific binders of the GFP/GFP-aptamer in human cells, but also in combination with the control RNA aptamer-enriched proteins from HSF1-GFP cells, allow us to calculate an enrichment factor for each identified protein and to identify high-confidence HSF1 interactors using the most stringent criteria (see Materials and methods). GO analysis of proteins enriched by the control aptamer shows that the vast majority of these proteins are devoted to binding nucleic acid. These nucleic acid-binding proteins are RNA binding proteins. It is predicted that these proteins are likely to bind RNA non-specifically, and proposed that they serve as a resource in RACER, analogous to the common contaminants repository used for antibody-based pull-downs (Mellacheruvu, et al., “The CRAPome: a Contaminant Repository for Affinity Purification-mass Spectrometry Data,” Nat. Methods 10:730-736 (2013), which is hereby incorporated by reference in its entirety). In addition, the non-specific proteins enriched from the GFP only cells by the GFP-aptamer also provide a list of contaminants for AptA-MS studies using GFP-fusions. RACER is publicly available and can be continually updated for use in further AptA-MS experiments.
Knock-in cell lines with GFP-tagged proteins generated by CRISPR/Cas9 are proving to be critical for imaging macromolecules in living cells (Steurer et al., “Live-cell Analysis of Endogenous GFP-RPB1 Uncovers Rapid Turnover of Initiating and Promoter-paused RNA Polymerase II,” Proc. Natl. Acad. Sci. U.S.A., 115:E4368-E4376 (2018), which is hereby incorporated by reference in its entirety). They also can be used directly for analysis by AptA-MS with the GFP aptamer, as this method is capable of purifying GFP and GFP-tagged proteins from various sources. Additionally, large libraries of GFP-tagged proteins are available, and any member of such libraries could also be used directly in AptA-MS, thereby allowing identification of the associated factors along with their PTMs in a single assay (Roberts, et al., “Systematic Gene Tagging Using CRISPR/Cas9 in Human Stem Cells to Illuminate Cell Organization,” Mol. Biol. Cell 28:2854-2874 (2017); Harikumar et al., “An Endogenously Tagged Fluorescent Fusion Protein Library in Mouse Embryonic Stem Cells,” Stem Cell Rep. 9:1304-1314 (2017), which are hereby incorporated by reference in their entirety). These findings would complement optical studies of cellular dynamics and co-localization with other proteins in vivo. The GFP-aptamer has been shown to bind to other derivatives of GFP, making it applicable to precipitate proteins tagged with similar fluorescent proteins (Shui et al., “RNA Aptamers that Functionally Interact with Green Fluorescent Protein and its Derivatives,” Nucleic Acids Res. 40:e39 (2012), which is hereby incorporated by reference in its entirety). Aptamers provide many advantages as affinity reagents: they can be selected against toxic proteins, are amenable for chemical modifications, are cost effective to synthesize, and can be made without the use of animals in any molecular biology lab in unlimited quantities. These advantages, in addition to its broad applicability, make AptA-MS a highly sensitive and simple tool that could significantly transform protein—protein interaction studies and provide deeper and more comprehensive insights in understanding the composition of MCs.
Lysate preparation. S. pombe strain used: ade6-m216, ura4-D18, leu1, gfp-rpb3 (Source: National Bio Resource Project, Japan). Lysate was prepared from 500 ml of culture (˜5 billion Yeast cells) expressing GFP-Rpb3 and was used for GFP-aptamer pulldown. Rpb3 is a subunit of RNA polymerase II (Pol II). Cells were cultured to OD600=1 (1.0×107 cells/ml) in 500 ml of YES media at 30° C. Cells were collected in 50 ml of culture into 50 ml conical tubes and centrifuged at 1100×g for 5 min. Medium was discarded and pellets were frozen in liquid N2 and stored at −20° C. Pellets were thawed by adding 1 ml of PBS and transferred into a 2 ml screw cap tube. Cells were centrifuged at 11,000×g for 5 min at 4° C. Pellets were resuspended in 800 μL of complete Lysis buffer (1× PBS+0.2% NP-40+1× PIC+ 1/50 0.1M PMSF). PIC=Protease inhibitor cocktail-EDTA free. Sample was centrifuged at 11,000×g for 5 min at 4° C. Supernatant was discarded and pellets were resuspended in 400 μL of complete Lysis buffer. Zirconia beads were added up to 80% of the 2 ml screw cap tube. Samples were Beadsbeaten by FastPres-24 (MB products) for 4 cycles with the settings below.
Tubes were punctured at the bottom of the 2 ml tube by a heated needle. The 2 ml tube was placed in a 7 ml polypropylene tube and centrifuged at 1100×g for 1 min at 4° C. The lysate pass through the puncture and was collected in the 7 ml tube. The crude lysate was transferred into a new 2 ml tube and centrifuge at 15,000×g for 10 min at 4° C. The supernatant was transferred to a 1.5 ml tube. Lysates were combine in one tube. And diluted 1:4 with Dilution buffer (1× PBS+250 ng/μL ssDNA+250 ng/μL yRNA+7 mM MgCl2).
RNA preparation and immobilization on beads. 100 pmole of polyadenylated GFP aptamer was annealed to desthiobiotin-oligodT-20 in 200 μL of 1× Annealing buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl) by heating at 95° C. for 3 min and slow cooling to room temperature over >1 hr. For pulldown 1 mg of Dynabeads MyOne Streptavidin C1 (Thermo) magnetic beads were washed once with 1 ml and twice with 0.1 ml of Tween wash buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1M NaCl, 0.05% Tween-20) by placing on a magnetic separator for 2 min and removing the supernatant. To eliminate possible RNase activity, beads were washed once with 0.1 ml of 0.1 M NaOH, 0.05 M NaCl followed by two washes of 0.1 ml of 0.1 M NaCl with changing tubes in between washes. The beads were resuspended in 200 μL of 2× Binding buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl) supplemented with 4 units/ml SUPERase IN. The resulting bead slurry was mixed with the annealed RNA aptamer and incubated on a thermomixer for 1 hr at 23° C. with shaking. Aptamer bound beads were washed twice with 0.4 ml of Bead wash buffer (1× PBS, 0.05% NP40, 5 mM MgCl2), supplemented with 4 units/ml SUPERase IN with changing tubes in between washes.
Binding to lysate, washing & elution. The GFP-aptamer bound beads were resuspended in the diluted cellular lysate and incubated at 4° C. with rotation for 2 hr. The beads were placed on a magnet and supernatant was removed. The beads were washed four times with Bead wash buffer and once with 1× PBS, 5 mM MgCl2, with changing tubes in between washes. Beads were resuspended in 50 μL of fresh elution buffer (5 mM Biotin, 50 mM Ammonium phosphate, pH 7.5) and incubated in a Thermomixer at 37° C. with shaking for 1 hr followed by collection of the eluate into a fresh tube.
Cryo-EM structure determination. The eluate was prepared for Cryo-EM analysis and the structure was determined. The electron micrograph of a graphene oxide grid below shows that the eluate was enriched in particles consistent with the expected size of Pol II (
Human HCT116 cells transfected with pEGFP of pHSF1-GFPN3 (Addgene #32538) plasmid were resuspended in 0.5 ml ice cold cellular lysis buffer (1× PBS+0.2% NP40+1× EDTA-free Protease inhibitor cocktail). Cells were incubated on ice for 30 min followed by sonication with Bioruptor Diagenode at High setting (30 s ON/30 s OFF) for 5 min. The lysate was centrifuged at 20,000×g for 10 min in 4° C. and the resulting supernatant was transferred to a new tube. The cleared lysate was diluted to a final buffer containing 1× PBS, 0.05% NP40, 5.25 mM MgCl2, 187.5 ng/μL yeast RNA, 187.5 ng/μL sheared salmon sperm DNA, 200 units SUPERase IN/ml.
RNA preparation and immobilization on beads. Around 200 pmole of polyadenylated GFP- or the control (NELF)-aptamer was annealed to equimolar Desthiobiotin-oligodT (20) in 200 μL of 1× Annealing buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl) by heating at 95° C. for 3 min and slow cooling to room temperature over >1 hr. For each pulldown 1 mg of Dynabeads MyOne Streptavidin C1 (Thermo) magnetic beads were washed once with 1 ml and twice with 0.1 ml of Tween wash buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1M NaCl, 0.05% Tween-20) by placing on a magnetic separator for 2 min and removing the supernatant. To eliminate possible RNase activity, beads were washed once with 0.1 ml of 0.1 M NaOH, 0.05 M NaCl followed by two washes of 0.1 ml of 0.1 M NaCl with changing tubes in between washes. The beads were resuspended in 200 μL of 2× Binding buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2M NaCl) supplemented with 4 units/ml SUPERase IN. The resulting bead slurry was mixed with the annealed RNA aptamer and incubated on a thermomixer for 1 hr at 23° C. with shaking. Aptamer bound beads were washed twice with 0.4 ml of Bead wash buffer (1× PBS, 0.05% NP40, 5 mM MgCl2), 4 units/ml SUPERase IN with changing tubes in between washes.
Binding to lysate, washing & elution. The GFP- or control-aptamer bound beads were resuspended in the diluted cellular lysate and incubated at 4° C. with rotation for 2 hr. The beads were placed on a magnet and supernatant was removed. The beads were washed twice with Bead wash buffer and twice with 1× PBS, 5 mM MgCl2, with changing tubes in between washes. Beads were resuspended in 50 μL of fresh elution buffer (5 mM Biotin, 50 mM Ammonium phosphate, pH 7.5) and incubated in a Thermomixer at 37° C. with shaking for 1 hr followed by collection of the eluate into a fresh tube.
DNA purification & library preparation for sequencing. Eluates were treated with 2 μL of RNase cocktail (RNase A/T1, Thermo) and incubated at 37° C. for 20 min. DNA was purified by Phenol/Chloroform, chloroform extraction followed by Ethanol precipitation and finally resuspended in 25 μL of 10 mM Tris-HCl, pH 8.5. Library was prepared by Tn5-tagmentation where 10 μL of DNA was treated with Tn5 reaction buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 10% DMF), 0.5 μL Tn5 complex with adaptors to a volume of 25 μL and incubated at 55° C. for 5 min. Reactions were quenched with 0.1% SDS and kept on ice. Tagmented DNA was PCR amplified with Nextera adaptors and Phusion Polymerase for 13 cycles. The amplicons were purified by Ampure beads and sequenced with pair-end sequencing using Illumina NextSeq500.
The GFP aptamer-based pulldown strategy could be implemented to identify DNA binding sites of a particular protein genome-wide. The aptamer pulldown methodology as described in
ChAP-seq clearly enriches for HSF1 binding sites genome-wide only in HSF1-GFP cells but not in the controls. Many of these peaks overlap with previously identified HSF1 peaks from ChlPseq data obtained from crosslinked human K562 cells. In summary, the aptamer pulldown strategy provides a simple, native method in which samples from a single precipitation technique can be processed for both AptA-MS as well as ChAP-seq thereby providing both protein and DNA interactor information from a single assay.
Human HCT116 cells expressing GFP-Rpb1 were grown in 15 cm dishes to confluency in McCoy's 5A Media+10% FBS+Penn/Strep. Media was removed and the cells were rinsed with 1× PBS. Cells were crosslinked with 0.5% formaldehyde for 5 min at room temperature with shaking. Crosslinking was quenched with 200 mM Glycine for 5 min at room temperature with shaking. Cells were collected and washed with 1× PBS. Cell pellets were flash frozen in liquid nitrogen and stored at −80° C. Crosslinked cell pellet was resuspended in 10 ml ice cold lysis buffer (10 mM Tris pH 8, 10 mM NaCl, 0.5% NP40+1× EDTA-free Protease inhibitor cocktail). Cells were incubated on ice for 15 min. Cells were spun at 500×g for 5 min at 4° C. and supernatant was removed. The pellet was resuspended in 10 ml ice cold lysis buffer and incubated on ice for 5 min. Cells were spun at 500×g for 5 min at 4° C. and supernatant was removed. Pellet was resuspended in 1× pellet volume of Nuclear lysis buffer (50 mM Tris pH 8+0.2% SDS) and transferred to a new tube. The tube was shaken in a thermomixer at room temperature for 5 min. Equal volume of SDS Quenching buffer (2× PBS+1% Triton X-100) was added. The suspension was divided into 1.5 ml tubes and sonicated with Bioruptor Diagenode at High setting (30 s ON/30 s OFF) for 15 min in ice cold water at 4° C. The lysate was centrifuged at 20,000×g for 10 min in 4° C. and the resulting supernatant was transferred to a new tube. The cleared lysate was diluted to a final buffer containing 1× PBS, 0.01% SDS, 0.1% Triton X-100, 5 mM MgCl2, 200 ng/μL yeast RNA, 0.1 mM Dextran Sulfate, 100 units SUPERase IN/ml.
RNA preparation and immobilization on beads. Around 225 pmole of polyadenylated GFP (C58U)- or the control (NELF)-aptamer was annealed to 200 pmole of DesthiobiotinoligodT (20) in 200 μL of 1× Annealing buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl) by heating at 95° C. for 5 min and slow cooling to room temperature over >1 hr. For each pulldown 0.5 mg of Dynabeads MyOne Streptavidin C1 (Thermo) magnetic beads were washed once with 0.5 ml and twice with 50 μL of Tween wash buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1M NaCl, 0.05% Tween-20) by placing on a magnetic separator for 2 min and removing the supernatant. To eliminate possible RNase activity, beads were washed once with 50 μL of 0.1 M NaOH, 0.05 M NaCl followed by two washes of 50 μL of 0.1 M NaCl with changing tubes in between washes. The beads were resuspended in 200 μL of 2× Binding buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2M NaCl) supplemented with 4 units/ml SUPERase IN. The resulting bead slurry was mixed with the annealed RNA aptamer and incubated on a thermomixer for 1 hr at 23° C. with shaking. Aptamer bound beads were washed twice with 0.4 ml of Bead wash buffer (1× PBS, 0.05% NP40, 5 mM MgCl2), 4 units/ml SUPERase IN with changing tubes in between washes.
Binding to lysate, washing & elution. The GFP- or control-aptamer bound beads were resuspended in the diluted lysate and incubated at 4° C. with rotation for 2 hr. The beads were placed on a magnet and supernatant was removed. The beads were washed with Wash buffer 1 (1× PBS, 0.1% NP40, 5 mM MgCl2, 0.1 mM DxSO4, SUPERase IN) twice with Wash buffer 2 (1× PBS, 0.1% NP40, 5 mM MgCl2, 200 mM NaCl, SUPERase IN) by shaking in a Thermomixer for 5 min at room temperature, once with Wash buffer 3 (1× PBS, 0.1% NP40, 5 mM MgCl2, SUPERase IN) and finally with 1× PBS, 5 mM MgCl2, with changing tubes in between washes. Beads were resuspended in 50 μL of fresh elution buffer (5 mM Biotin, 50 mM Ammonium phosphate, pH 7.5, 4 μL RNase A/T1 cocktail) and incubated in a Thermomixer at room temperature with shaking for 30 min followed by collection of the eluate into a fresh tube.
DNA purification, qPCR, & sequencing. DNA was purified by Qiagen Minelute Reaction cleanup kit. qPCR was performed with target site (GAPDH primers from Erickson et al, Genes &. Dev (2018), which is hereby incorporated by reference in its entirety) and non-target site primers (2 Kb upstream of GAPDH transcription start site) using Roche Lightcycler qPCR instrument.
For DNA sequencing the library was prepared by Tn5-tagmentation where 10 μL of DNA was treated with Tn5 reaction buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 10% DMF), 0.5 μL Tn5 complex with adaptors to a volume of 25 μL and incubated at 55° C. for 5 min. Reactions were quenched with 0.1% SDS and kept on ice. Tagmented DNA was PCR amplified with Nextera adaptors and Phusion Polymerase for 13 cycles. The amplicons were purified by Ampure beads and sequenced with pair-end sequencing using Illumina HiSeq system.
It is shown that the GFP-aptamer can be utilized to purify a protein of interest from native cellular lysates and identify the DNA sequences bound to the protein by Next generation sequencing. Here, this Chromatin-Aptamer Precipitation (ChAP) strategy was further implemented to identify DNA-binding sites of a particular protein of interest from formaldehyde (FA) crosslinked cells, an extended method that is referred hereby as FA-ChAP (
FA-ChAP provides a highly specific approach to enrich and identify DNA sequences bound to a GFP-tagged protein of interest upon formaldehyde fixation. Formaldehyde has been used for decades to capture transient macromolecular interactions of many proteins including the transcription factors that are loosely bound to the underlying DNA. Standard Chromatin immunoprecipitation (ChIP) utilizes formaldehyde to crosslink the cells before performing the immunoprecipitation step in order to stabilize the DNA-protein complex. Although many antibodies perform well for native detection/precipitation methods, all are not suited for ChIP assays. The FA-ChAP results demonstrate that the GFP-aptamer is highly suitable for precipitating a GFP-tagged protein even upon formaldehyde crosslinking. The high signal-noise ratio detected by DNA sequencing of the pulled down material makes FA-ChAP an inexpensive and rapid method to identify the DNA binding sites of the tagged protein with high sensitivity, thereby allowing identification of potential novel binding sites.
The idea of a sequential affinity-purification where two proteins (one of them being GFP-tagged) co-occupying a sequence of DNA can be purified by their respective affinity reagents and the underlying DNA can be isolated and analyzed is shown in
The same idea could be reversed where the first capture is done with the antibody and the second one is done with the GFP-aptamer (
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the present application and these are therefore considered to be within the scope of the present application as defined in the claims which follow.
This application claims the benefit of U.S. Provisional Patent Application Ser. Nos. 62/970,035 and 62/970,015, filed Feb. 4, 2020, which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62970015 | Feb 2020 | US | |
| 62970035 | Feb 2020 | US |
