Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
  • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding

Definitions

• the invention relates to a fusion protein with applications in cell labeling, nanopore sequencing and the organizing of cells.
• a second set of unsolved problems have hampered the development of nanopore sequencing. These critical technical challenges include the need for a means to guide the polymer through the nanopore and a means to regulate the rate at which the polymer passes through.
• the present invention seeks to address these problems.
• the present invention uses polynucleotide -binding domains and complementary polynucleotides to provide solutions to the above-mentioned challenges.
• DNA binding domains such as zinc finger proteins (ZFs) and more recently transcription activator- like effectors (TALEs) and their ligands provide a unique and versatile tool, since both the receptor (ZF or TALE protein) and the ligand (DNA) are highly programmable and hence the space of orthogonal interactions that can be engineered is potentially infinite.
• the invention relates to a protein including (a) a polynucleotide- binding domain; and (b) a transmembrane domain.
• the polynucleotide -binding domain is a programmable DNA binding domain.
• the polynucleotide-binding domain is a zinc finger protein or TAL effector protein.
• the zinc finger domain is a C2H2 triple finger.
• the zinc finger is a synthetic zinc finger such as a zinc finger selected from the group consisting of Zl, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Zl 1, Z12, Z13, Z14, Z15, and Z16.
• the protein includes an Ig ⁇ -chain leader sequence at its N-terminus.
• the protein includes an import sequence at its C- terminus.
• the transmembrane domain is C-terminal of the polynucleotide-binding domain.
• the protein is a nanopore subunit.
• the invention relates to a polynucleotide encoding the above-mentioned protein.
• the polynucleotide may be operably linked to a promoter.
• the polynucleotide is a free, linear unit.
• the invention relates to a plasmid including the polynucleotide.
• the invention relates to a viral vector including the polynucleotide.
• the invention relates to a cell including the abovementioned polynucleotide, plasmid, or vector.
• the invention relates to a protein complex including one or more proteins of the first aspect.
• the complex is a nanopore.
• the nanopore is selected from the group consisting of alpha-hemolysin (aHL) and mycobacterium smegmatis porin A (MspA).
• aHL alpha-hemolysin
• MspA mycobacterium smegmatis porin A
• the invention relates to a membrane including one or more variants of the nanopore.
• the invention relates to a cell including one or more variants of the protein or protein complex, where the polynucleotide-binding domain(s) of the protein(s) or protein complex(es) are on the surface of the cell in a manner capable of interaction with materials external to the cell.
• the invention in another aspect, relates to a method of sequencing a polynucleotide, the method including: (a) providing two separate pools of liquid containing electrically conducive medium and a nanopore-perforated interface between the two pools, where the interface contains a nanopore; (b) providing candidate polynucleotide molecules in one of the pools; (c) applying a voltage differential across the pools; and (d) making interface-dependent measurements of ionic current over time as individual nucleotides of a single polynucleotide interact sequentially with the interface, yielding data suitable to determine a nucleotide-dependent characteristic of the polynucleotide.
• the interface is a membrane.
• the invention in another aspect, relates to a method of tagging a cell including the steps of (a) providing a cell including one or more variants of the protein or protein complex, where the polynucleotide-binding domain(s) of the protein(s) or protein complex(es) are on the surface of the cell in a manner capable of interaction with materials external to the cell; (b) providing a polynucleotide probe including a detectable label and capable of interacting with the surface polynucleotide-binding domain(s); (c) contacting the cell with the polynucleotide probe; and (d) detecting the polynucleotide probe.
• the detectable label is a fluorescent label or a luminescent label, quenching label, biotin label, ligation label, chemically reactive moiety such as azide, chemically reactive moiety that enables click-chemistry, detectable small molecule, or the sequence of polynucleotide probe.
• the invention in another aspect, relates to a method of labeling and sequentially relabeling cells, the method including the steps: (a) providing a cell including one or more variants of the protein or protein complex, where the polynucleotide-binding domain(s) of the protein(s) or protein complex(es) are on the surface of the cell in a manner capable of interaction with materials external to the cell; (b) providing a polynucleotide probe including a detectable label that is capable of interacting with the surface polynucleotide-binding domain(s); (c) contacting the cell with the polynucleotide probe; (d) detecting the polynucleotide probe; (e) dissociating the polynucleotide probe from the polynucleotide-binding domain(s); (f) providing a subsequent polynucleotide probe including a detectable label; (g) contacting the cells with the subsequent polynucleotide probe; and (h) detecting the
• one or more of the detectable label is a fluorescent label or a luminescent label, quenching label, biotin label, ligation label, chemically reactive moiety such as azide, chemically reactive moiety that enables click-chemistry, detectable small molecule, or the sequence of polynucleotide probe.
• the dissociation is achieved by the addition of a high concentration of unmarked polynucleotide(s) capable of displacing the polynucleotide probe.
• the method further includes repeating one or more times the steps (e) to (h) with an additional polynucleotide probe or probes.
• the invention in another aspect, relates to a method of labeling a cell, the method including the steps of (a) providing a cell providing a cell including one or more variants of the protein or protein complex, where the polynucleotide-binding domain(s) of the protein(s) or protein complex(es) are on the surface of the cell in a manner capable of interaction with materials external to the cell; (b) providing a scaffold polynucleotide to mediate the interaction of the surface polynucleotide biding domain(s) with one or more polynucleotide probe(s), the scaffold polynucleotide including (i) a double-stranded domain and (ii) a single-stranded domain including one or more sites capable of hybridization, where the double-stranded domain interacts with the surface polynucleotide-binding domain(s) and the single- stranded domain is available to interact with one or more polynucleotide probes; (c) providing a
• the detectable label is a fluorescent label or is a luminescent label, quenching label, biotin label, ligation label, chemically reactive moiety such as azide, chemically reactive moiety that enables click- chemistry, detectable small molecule, or the sequence of polynucleotide probe.
• the invention in another aspect, relates to a method of labeling and sequentially relabeling cells, the method including the steps of (a) providing a cell providing a cell including one or more variants of the protein or protein complex, where the polynucleotide-binding domain(s) of the protein(s) or protein complex(es) are on the surface of the cell in a manner capable of interaction with materials external to the cell; (b) providing a scaffold polynucleotide to mediate the interaction of the surface polynucleotide biding domain(s) with one or more polynucleotide probe(s), the scaffold polynucleotide including (i) a double-stranded domain and (ii) a single-stranded domain including one or more sites capable of hybridization, where the double-stranded domain interacts with the surface polynucleotide-binding domain(s) and the single-stranded domain is available to interact with one or more polynucleotide probes; (c)
• the labels are fluorescent labels and the mechanism of quenching is a mechanism of quenching fluorescence.
• one or more of the detectable labels is a luminescent label, quenching label, biotin label, ligation label, chemically reactive moiety such as azide, chemically reactive moiety that enables click-chemistry, detectable small molecule, or the sequence of said polynucleotide probe.
• the method further includes the step of (k) repeating one or more times the steps (f) to (j) with additional quenching polynucleotide(s) and polynucleotide probe(s).
• the cells are fixed or are growing adherently. In some embodiments, the cells are in solution.
• expression of the polynucleotide- binding domain is under control of a promoter and the level of labeling is indicative of promoter activity.
• the invention in another aspect, relates to a method for organizing cells, the method including the steps of (a) providing a solid surface to which one or more polynucleotides are affixed in a defined manner; (b) providing a cell or cells including a surface polynucleotide-binding domain capable of directly binding the affixed polynucleotides; and (c) contacting the solid surface with the cells, thereby affixing the cells.
• the affixed cells include an additional surface polynucleotide-binding domain
• the method further includes the steps : (d) providing a new cell or cells including surface polynucleotide-binding domains; (e) providing one or more polynucleotide(s) capable of tethering the affixed and the new cells in a sequence-specific manner; and (f) contacting the tethering polynucleotide(s) with the affixed cells and the new cells.
• the tethering polynucleotide is a double-stranded polynucleotide probe including regions capable of being bound by the surface polynucleotide-binding domains of both the affixed cells and the new cells.
• the tethering is mediated by two polynucleotides, each including (i) a double-stranded domain capable of interacting with a surface polynucleotide-binding domain and (ii) a single-stranded domain, and where one tethering probe's double-stranded domain interacts directly with the affixed cells, the other tethering probe's double-stranded domain interacts directly with the new cells, and the single- stranded domains of the two tethering probes are complementary so as to tether the cells.
• the method further includes the step: (h) repeating steps (d)- (f) one or more times with additional cells and/or tethering polynucleotides.
• the invention in another aspect, relates to a method for organizing cells, the method including the steps of (a) providing a solid surface to which one or more polynucleotides are affixed in a defined manner; (b) providing a cell or cells including a surface polynucleotide-binding domain; (c) providing a scaffolding polynucleotide capable of binding the cell surface polynucleotide-binding domain, the polynucleotide probe including (a) a double-stranded domain and (b) a single-stranded domain, where the double-stranded domain interacts with the surface polynucleotide-binding domain(s) and the single-stranded domain is available to interact with one or more of the affixed polynucleotides; and (d) contacting the solid surface with the cells, thereby affixing the cells.
• the affixed cells include an additional surface polynucleotide-binding domain
• the method further includes the steps of (e) providing a new cell or cells including surface polynucleotide-binding domains; (f) providing one or more polynucleotide(s) capable of tethering the affixed and the new cells in a sequence-specific manner; and (g) contacting the tethering polynucleotide(s) with the affixed cells and the new cells.
• the tethering polynucleotide is a double-stranded polynucleotide probe including regions capable of being bound by the surface polynucleotide-binding domains of both the affixed cells and the new cells.
• the tethering is mediated by two polynucleotides, each including (i) a double-stranded domain capable of interacting with a surface polynucleotide-binding domain and (ii) a single- stranded domain, and where one tethering probe's double-stranded domain interacts directly with the affixed cells, the other tethering probe' s double-stranded domain interacts directly with the new cells, and the single-stranded domains of the two tethering probes are complementary so as to tether the cells.
• the method further includes the step of (h) repeating steps (e)-(g) one or more times with additional cells and/or tethering polynucleotides.
• the invention in another aspect, relates to a method of tethering free cells, the method including the steps of (a) providing two or more free cells including one or more surface polynucleotide-biding domains; (b) providing one or more tethering polynucleotides capable of tethering two of the free cells; and (c) contacting the one or more polynucleotides with the free cells.
• the tethering polynucleotide is a double-stranded polynucleotide probe including regions capable of being bound by the surface polynucleotide-binding domains of both the affixed cells and new cells.
• the tethering is mediated by two polynucleotides, each including (i) a double-stranded domain capable of interacting with a surface polynucleotide- binding domain of the free cells and (ii) a single-stranded domain, where the single- stranded domains of the two tethering probes are complementary so as to tether the free cells.
• the method further includes the step of (d) Repeating steps (a)-(c) one or more times with additional cells and/or tethering polynucleotides.
• the invention relates to an artificial tissue produced by the above-mentioned methods.
• the artificial tissue is vascular tissue or an organoid.
• Fusion protein means a protein having a sequence derived from two or more source proteins.
• Fusion gene means a gene that encodes a fusion protein.
• Polynucleotide -binding domain means any polypeptide or portion of a polypeptide that binds polynucleotides with specificity toward polypeptides of a particular sequence, sequences, or class of sequences.
• DNA binding domain means a polynucleotide-binding domain that interacts with DNA.
• Surface polynucleotide-binding domain means a polynucleotide-binding domain integrated within the membrane of a cell in a manner that permits the interaction with polynucleotides external to the cell.
• Nanopore means a hole or passage through a membrane formed by a multimeric protein ring. Typically, the passage is 0.2-25 nm wide.
• Nanopore sequencing means a method of determining the components of a polymer, such as a polynucleotide, based upon interaction of the polymer with the nanopore. Nanopore sequencing may be achieved by measuring a change in the conductance of ions through a nanopore that occurs when the size of the opening is altered by interaction with the polymer.
• Polynucleotide probe means any polynucleotide that may be appended to a cell in a manner that permits the detection of the cell, assignment of identity or class of the cell, or enables the utilization of the cell for a desired purpose.
• a probe may include a detectable label.
• Tag means any process that results in the appending of a probe to a cell.
• “Scaffold polynucleotide” means a polynucleotide probe that includes a double stranded domain and a single stranded domain, such that the double stranded domain is designed to interact with a surface polynucleotide-binding domain and the single stranded domain is designed to interact with one or more polynucleotide probes to guide the association of these elements.
• tissue means any group of cells organized into a defined structure.
• Figure 1 is a table listing 16 synthetic zinc fingers with their protein sequences, target sequences, and the vectors constructed.
• Figure 2 is a schematic presenting one non- limiting mechanism by which cells expressing fusion polynucleotide -binding proteins might express them at the cell surface and interact with complementary DNA molecules.
• Figure 3 is a series of eight images (2 phase and 6 fluorescence) showing that zinc finger fusion proteins expressed in cells are able to bind complementary probes with specificity.
• Figure 4a is a set of four images showing the interaction of cells expressing a zinc finger fusion protein with a dsDNA or ssDNA probe in the presence or absence of salmon sperm DNA.
• Figure 4b is a set of 12 images showing the interaction of cells expressing a zinc finger fusion protein with a dsDNA probe in the presence or absence of an ssDNA probe.
• Figure 4c is 4 graphs displaying a
• Figure 5a is a set of 3 images showing cells expressing two zinc finger fusion proteins in the presence of complementary dsDNA probes and a scatter plot showing the overlap characteristics of the two probes.
• Figure 5b is two images showing the labeling of cells expressing two or three zinc finger fusion proteins and two scatter plots showing overlap when labeling the zinc fingers with complementary probes.
• Figure 5c is 4 images of cells expressing one, two or three zinc finger fusion proteins in a total of 7 combinations and labeled with each of three probes, with an overlay displayed in the fourth image.
• Figure 6a shows a series of 16 images, each individually showing a cell expressing a zinc finger fusion protein in the presence of complementary dsDNA probe.
• Figure 6b shows cells expressing each of 16 zinc finger fusion proteins and the interaction of each when probed with 16 probes.
• Figure 7 is a schematic workflow of an image analysis strategy that may be employed to analyze the binding of probes to polynucleotide -binding domains.
• Figure 8 is a bar graph showing the correlation of fluorescence intensity of identified cell regions.
• Figure 9a is a chart showing the results of image analysis, including the counts and fractions of segments and NOVS formed per channel aggregated over all images and all channels.
• Figure 9b is a chart showing the results of image analysis, including overall segment and NOVS counts and fractions form each channel in each image.
• Figure 10 is a pair of box plot charts showing the segment area size distribution for all images analyzed.
• Figure 1 1 is a box plot chart and a bar graph showing segment intensity distribution of all images analyzed.
• Figure 12a shows 3 scatter plots and 3 pixel histograms showing, respectively, the mean intensities resulting from image analysis and the pixel counts resulting from image analysis.
• Figure 12b shows 3 scatter plots and 3 pixel histograms showing, respectively, the mean intensities resulting from image analysis and the pixel counts resulting from image analysis.
• Figure 13 shows 5 scatter plots and 15 pixel histograms showing, respectively, the mean intensities resulting from image analysis and the pixel counts resulting from image analysis.
• Figure 14a shows 2 sets of 5 images, each set displaying a 48 minute time course in which cells expressing a zinc finger fusion protein in the presence of a complementary dsDNA probe are treated with either salmon sperm DNA or salmon sperm DNA and a high concentration of a non- fluorescent target dsDNA in solution.
• Figure 14b is a chart displaying the residual mean normalized pixel intensities measured over the 48 minute time course of dissociation.
• Figure 15a is a schematic presenting one non- limiting method by which cells expressing fusion polynucleotide-binding proteins might be sequentially labeled and re-labeled through the use of quencher probes.
• Figure 15b is a series of images showing the labeling, quenching, and subsequent relabeling of cells expressing one of three zinc finger fusion proteins and a schematic showing a labeling scheme for the zinc finger fusion protein-expressing cells utilized in the images, as well as three others.
• Figure 15c is (a) a schematic presenting one non- limiting method by which cells expressing fusion polynucleotide-binding proteins can be sequentially labeled and re-labeled through the use of quencher probes and (b) 4 images showing mixed cells, each expressing one of 6 zinc finger fusion proteins, that are labeled, quenched, and re-labeled.
• Figure 16 is a schematic presenting one non- limiting method by which a zinc finger fusion protein could serve as a reporter of endogenous activity and a series of 12 images showing cells with an inducible zinc finger fusion protein in the presence of a complementary probe under conditions that induce or do not induce expression of the zinc finger fusion protein.
• Figure 17 is a set of 4 images showing cells with an inducible zinc finger fusion protein in the presence of a complementary probe under conditions that induce or do not induce expression of the zinc finger fusion protein.
• the present invention relates to a fusion protein comprising a polynucleotide-binding domain and a transmembrane domain.
• the fusion protein is expressed at the cell surface and is thereby available to bind polynucleotides outside the cell.
• the applications of this protein include the improvement of nanopore technology for DNA sequencing, advanced methodologies for labeling cells, and construction of artificial tissues.
• the polynucleotide-binding domain of the fusion protein may be any polypeptide that is known to bind polynucleotides, such as DNA, or that is encompassed by a family of proteins known to bind polynucleotides.
• Zinc fingers are a well-recognized group of polynucleotide-binding proteins, encompassing various subclasses of fold groups including Cys 2 His 2 , gag knuckle, treble clef, zinc ribbon, Zn 2 /Cys 6 , and TAZ2 domain-like factors.
• polynucleotide-binding domains include helix-turn-helix, leucine zipper, winged helix, winged helix-turn-helix, helix-loop-helix, HMG-box, immunoglobulin fold, B3 domain, TAL effector DNA-binding domain, and other domains. These and others known in the art are suitable for use in the current invention.
• Polynucleotide-binding proteins like zinc fingers can be modified to generate polynucleotide-binding proteins that bind a desired sequence or class of sequences with specificity.
• the Cys 2 His 2 motif is especially well-suited to such modification, providing a scaffold upon which specificity factors can be assembled by methods that will be familiar to those skilled in the art.
• a transmembrane domain is another component of the fusion protein of present invention.
• the transmembrane domain may be any three-dimensional structure which is thermodynamically stable in a membrane.
• the transmembrane domain may be a single transmembrane alpha helix.
• the transmembrane domain can be a helical bundle, seven-transmembrane protein, beta barrels, or any other class of transmembrane domain known in the art, as well as any member of such a class or any polypeptide otherwise known to act as a transmembrane domain.
• the transmembrane domain is C-terminal of the polynyucleotide-binding domain.
• a transmembrane domain is defined as a polypeptide sequence with high hydrophobicity score based on any of the algorithms known in the art or with a long series of hydrophobic residues.
• the transmembrane domain is a platelet derived growth factor (PDGF) transmembrane domain.
• PDGF platelet derived growth factor
• the transmembrane domain is selected from the transmembrane regions of transmembrane proteins of known structure or related proteins.
• Transmembrane proteins of known strucutre include ram prostaglandin H2 synthase- 1 (COX-1), squalene-hopene cyclase: Alicyclobacillus acidocaldarius, monoamine oxidase B: human mitochondrial outer membrane, fatty acid amide hydrolase: Rattus norvegicus, sulfide :quinone oxidoreductase in complex with decylubiquinone: Aquifex aeolicus, porin: Rhodobacter capsulatus, TolC outer membrane protein: Escherichia coli, bacteriorhodopsin (BR): Halobacterium salinarium, and many others, as curated in a list available at [http://blanco.biomol.uci.edu/membrane_proteins_xtal.html], the contents of which is herein incorporated by reference.
• COX-1 ram prostaglandin H2 synthase-
• the transmembrane domain is selected from the transmembrane regions of transmembrane proteins known in humans or related proteins, as curated in a list available at [https://modbase.compbio.ucsf.edu/projects/membrane/] and incorporated herein by reference.
• the invention features any genetic material that may be translated into the fusion gene of the invention.
• the protein may be expressed from a linear segment of DNA, DNA encoded within a plasmid, DNA encoded within a virus, DNA incorporated within a genome, DNA present within a cell, or DNA present within an organism.
• the fusion protein may be part of a nanopore or nanopore subunit.
• Nanopores are transmembrane structures that may permit the passage of molecules through a membrane.
• a multimeric nanopore may include any number of the fusion nanopore subunits (such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 fusion nanopore subunits) and any number of other subunits (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 other nanopore subunits).
• Examples of nanopores include a- hemolysin (Staphylococcus aureus) and MspA (Mycobacterium smegmatis).
• the nanopore fusion gene comprises or is part of an a-hemolysin or MspA multimer.
• the nanopore fusion gene comprises or is part of a pore-forming toxin, such as the ⁇ - PFTs Panton-Valentine leukocidin S, aerolysin, and Clostridial Epsilon-toxin, the a-PFTs cytolysin A, the binary PFT anthrax toxin, or others such as pneumolysin or gramicidin.
• a pore-forming toxin such as the ⁇ - PFTs Panton-Valentine leukocidin S, aerolysin, and Clostridial Epsilon-toxin
• the a-PFTs cytolysin A the binary PFT anthrax toxin
• others such as pneumolysin or gramicidin.
• Nanopores have become technologically and economically significant with the advent of nanopore sequencing technology.
• Methods for nanopore sequencing are known in the art, for example, as described in U.S.P.N. 5,795,782, which is incorporated by reference.
• nanopore sequencing involves a nanopore-perforated membrane immersed in a voltage-conducting fluid. A voltage is applied across the membrane, and an electric current results from the conduction of ions through the nanopore.
• the nanopore interacts with polymers, such as DNA, flow through the nanopore is modulated in a monomer-specific manner, resulting in a change in the current that permits identification of the monomer(s).
• the development of nanopore sequencing is hampered by critical technical challenges, including the need for a means to guide the polymer through the nanopore and a means to regulate the rate at which the polymer passes through.
• the present invention provides a solution to these challenges by placing a polynucleotide-binding domain at the nanopore to modulate the movement of the polymer.
• the polynucleotide-binding domain of the nanopore fusion gene binds polynucleotides and guides their progress through the nanopore at a moderated rate.
• the fusion nanopore can be integrated with other nanopore technologies.
• a DNA polymerase is also used to ratchet a DNA substrate through the nanopore.
• the polymerase is phi29 DNA polymerase, T7 DNA polymerase, Klenow fragment of DNA polymerase 1 , or other DNA polymerase.
• passive approaches can be used to slow the movement of DNA. These approaches may include nucleotide labeling, end termination of ssDNA with DNA hairpins, the use of positively charged residues in the nanopore as molecular brakes, and modification of pore shape to optimize processivity.
• the polynucleotide-binding domain of the fusion nanopore is non- sequence-specific and interacts with polynucleotides with minimal or no sequence discrimination. In some embodiments, a non-specific polynucleotide-binding domain is used for sequencing.
• the polynucleotide-binding domain of the fusion nanopore is specific to particular polynucleotide sequences or classes of sequences.
• a specific polynucleotide-binding domain is used, for instance, for use in polymorphism analysis, analysis of particularly selected loci, medical diagnostic applications, forensics, or other purposes.
• the membrane of the nanopore sequencing technique is lipid bilayer. In some embodiments, the membrane of the nanopore sequencing technique is an artificial membrane, composed of a material such as A1 2 0 3 , Ti0 2 , Hf0 2 , Si0 2 , SiN, or grapheme.
• the various embodiments of the fusion nanopore may be employed in the sequencing of single-stranded or double-stranded DNA, cDNA, RNA, mRNA, tRNA, rRNA, microRNA, siRNA, or any polynucleotide, as well as other polymers including but not limited to polypeptides.
• the fusion protein is expressed within or on the surface of a cell.
• the fusion protein is associated with the membrane of a cell in a manner that permits the polynucleotide-binding domain to interact with polynucleotides external to the cell.
• the fusion protein includes a secretion signal to direct the fusion protein to the cell surface.
• One such secretion signal is the IgK-chain leader sequence.
• an IgK- chain leader sequence is at the N-terminus of the fusion protein.
• the fusion protein includes an endoplasmic reticulum import sequence to direct the fusion protein to the cell surface. An endoplasmic reticulum import sequence may be based upon the serotonin receptor 5HT3A.
• a surface polynucleotide -binding protein is used as part of a cell- labeling strategy.
• a shortfall of fluorescent labeling, a primary labeling method in the art, is that it relies upon the detection of a limited number or colors.
• surface polynucleotide-binding proteins enable a barcoding strategy capable of distinguishing a much greater number of cells or cell types.
• each cell or cell type of interest expresses or is engineered to express one or more distinct surface polynucleotide-binding domain(s) with specificity to a unique polynucleotide sequence.
• the cells may then be contacted with polynucleotide probes, some or all of which are complementary to the polynucleotide-binding domain(s) expressed by the population of cells.
• the probes can interact with the polynucleotide-binding domains, tagging the cells.
• the probes may be labeled.
• the probes are labeled with fluorescent moieties.
• each probe is labeled with a distinct fluorescent moiety.
• the probes tag the cells expressing the complementary polynucleotide-binding domain(s), and cells expressing each probed polynucleotide-binding domain are then recognizable by fluorescent microscopy.
• the number of distinguishable fluorescent moieties is limited, the number of species distinguishable by this method alone is limited.
• the initial set of probes is dissociated. This dissociation can be achieved by the addition of a high concentration of unlabeled probes targeted to the same polynucleotide-binding domain(s) as the initial probes, displacing the initial probes.
• a subsequent, distinct probe set may then be introduced, relabeling the cells.
• the subsequent probe set targets a distinct, previously un- targeted set of polynucleotide-binding domain(s) expressed by the same set of cells.
• each of the subsequent probes is labeled with a distinct fluorescent moiety.
• the probes then tag the cells expressing the complementary polynucleotide-binding domain(s), and the identity of cells expressing each probed polynucleotide-binding domain is recognizable by fluorescent microscopy. Because the color coding of the second probe set is independent of the first, the number of cells or cell types that can be distinguished increases exponentially with each subsequent round of labeling.
• the relabeling of the cells may be repeated indefinitely. In one embodiment, relabeling is carried out until all cell types have been distinguished or the availability of unique polynucleotide- binding domain(s) has been exhausted.
• the series of labels associated with each cell over the course of one or more rounds of labeling serves as a barcode for the identity of that cell, as defined by the surface polynucleotide -binding domains it expresses.
• cells are fixed or growing adherently.
• the cells are in solution, such as liquid media.
• one or more polynucleotide probes are prehybridized with an oligonucleotide.
• labels or methods of probe detection may include, but need not be limited to, fluorescence, quenching, luminescence, ligation, PCR, chemically re-active moieties such as azides that enable click-chemistry, biotinylation and others.
• probes associate indirectly with the surface polynucleotide- binding domains.
• a scaffold polynucleotide binds the surface polynucleotide-binding domain, and the probes then bind the scaffold. Because a single scaffold can bind multiple probes, this method allows barcoding without dissociation steps and requires only one surface polynucleotide-binding domain per cell type.
• a cell or cell type of interest expresses or is engineered to express one or more distinct surface polynucleotide-binding domain(s) with specificity to a unique polynucleotide sequence.
• the cells may then be contacted with one or more scaffold polynucleotides.
• Each scaffold polynucleotide includes both a double-stranded domain and a single-stranded domain.
• the double- stranded domain interacts with the surface polynucleotide-binding domain(s) to which it is complementary, while the single-stranded domain remains available for interaction with additional reagents, such as polynucleotide probes.
• each distinct scaffold polynucleotide bears a distinct single-stranded domain.
• cells are then contacted with polynucleotide probes complementary to the available single- stranded domains of the bound scaffold polynucleotides.
• the length of the single- stranded domain of the scaffold polynucleotide may be greater than the length of the polynucleotide probe, such that a single scaffold single-stranded domain could accommodate multiple polynucleotide probes.
• the probes may be labeled.
• the probes will be labeled with fluorescent moieties.
• each probe will be labeled with a distinct fluorescent moiety.
• the probes can tag the cells associated with the complementary single-stranded domain(s), and surface polynucleotide-binding domains of each cell are then visualized by fluorescent microscopy.
• the label of the initial probe may be quenched by a signal-quenching polynucleotide.
• the signal-quenching probe is a polynucleotide that is complementary to a region adjacent to that bound by the initial probe and that bears a signal-quenching mechanism.
• the label is a fluorescent label and the signal-quenching mechanism is a fluorescence- quenching mechanism.
• cells are contacted with these signal-quenching probes and the signal generated by the label of the initial probe is extinguished. The cells are thereby primed for subsequent re-labeling.
• cells are subsequently probed with a second set of labeled polynucleotide probes.
• these probes are designed to target a segment of the single-stranded domain of the scaffold polynucleotide that remains unbound by either the initial probe or the signal-quenching probe.
• the process of labeling, quenching, and relabeling may followed by a second quenching and, in some embodiments, repeated in sequence until all cells are distinguished or all probe binding sites are exhausted.
• the labels are fluorescent labels and the signal-quenching mechanism is a fluorescence-quenching mechanism.
• cells are fixed or growing adherently.
• the cells are in solution, such as liquid media.
• one or more polynucleotide probes are prehybridized with an oligonucleotide.
• labels or methods of probe detection may include, but need not be limited to, fluorescence, quenching, luminescence, ligation, PCR, chemically re-active moieties such as azides that enable click-chemistry, biotinylation and others.
• the present invention provides tools for the determination of gene expression.
• the expression of a fusion gene of the present invention is driven by the promoter of a gene of interest. In this way, expression of the fusion gene is indicative the transcriptional activity of the gene of interest.
• the promoter of the gene of interest drives expression of the fusion gene.
• the fusion gene is expressed in tandem with the entire gene of interest, or a portion thereof.
• the protein translated from the expressed fusion gene associates with the cell membrane and presents a surface polynucleotide-binding domain.
• the polynucleotide-binding domain may be labeled by any of the direct or indirect labeling methods described above, and the level of labeling is indicative of the activity of the utilized promoter.
• polynucleotide probes are affixed to a solid surface in a defined manner. These polynucleotide probes are complementary to one or more surface polynucleotide- binding domains present on a provided set of cells. Upon contacting the cells with the affixed polynucleotides, the surface polynucleotide biding domains bind the probes to which they are complementary, thereby forming a layer of cells organized according to the interaction of the fusion gene and the probes. In some embodiments, the cells possess unbound surface polynucleotide-binding domains available to interact with additional polynucleotides.
• a second set of cells is organized upon the first.
• the surface-affixed cells are contacted with a second set of cells with surface polynucleotide-binding domains and a polynucleotide tether.
• the polynucleotide tether is a double-stranded polynucleotide with ends respectively complementary to surface polynucleotide-binding domains present on the first or second set of cells.
• the tether may composed of two scaffold polynucleotides, with double-stranded regions complementary to the surface polynucleotide-binding domains of the initial or new cells, respectively, and single-stranded domains complementary to each other.
• the specificity of the surface polynucleotide-binding domains defines the manner in which the second set of cells is arranged upon the first.
• the initial set of cells may be tethered to the surface- affixed probes by a scaffolding polynucleotide having a double-stranded domain complementary to one or more surface polynucleotide-binding domains on the cells and a single- stranded domain complementary to the surface-affixed probes.
• similar strategies may be employed to tether cells in solution, such as liquid media.
• Two or more cells, each expressing one or more surface polynucleotide-binding domains, could be tethered directly by a double-stranded polynucleotide tether or indirectly by a pair of scaffolding polynucleotides with complementary single-stranded domains.
• subsequent cells and polynucleotides could be added to generate tissues of increasing complexity.
• one or more polynucleotide probes, scaffolds or tethers are prehybridized with an oligonucleotide.
• the number of distinct probes, scaffolds, tethers and cell types added in each step may be limited (such as 1, 2, or 3) or extensive (such as 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000).
• the subsequent tethering of additional sets of cells may be carried out repeatedly as described, generating tissues of increasing complexity.
• tissues that may be constructed, or of which models or equivalents may be constructed, in the described fashion include, but are not limited to organoids, vascular networks, muscle tissue and any tissue found in the human body.
• Zinc finger proteins expressed on the cell membrane can bind their cognate target dsDNA specifically in both simplex and multiplex settings and can thus serve as faithful cellular barcodes.
• fluorescent proteins like fluorescent proteins they can also serve as reporters of endogenous activity such as transcriptional activation. This feature opens the possibility to doing highly-multiplexed tracking of endogenous genes and pathways.
• This feature opens the possibility to doing highly-multiplexed tracking of endogenous genes and pathways.
• an almost arbitrary number of thus barcoded cells in complex mixtures can be imaged.
• sZFs are expressed on the cell surface they are physically accessible and hence offer the ability to not only label cells using DNA probes (as outlined above), but they also provide convenient handles for DNA mediated cell capture, and targeting of barcoded cells. Thus as compared to fluorescent proteins or antibodies, sZFs provide more versatile barcodes for cells.
• Example 1 Design and construction of synthetic polynucleotide -binding domain/polynucleotide pairs DNA binding domains such as zinc finger proteins (ZFs), and more recently transcription activator- like effectors (TALEs), have found numerous applications as synthetic transcription factors and editing tools such as site-specific nucleases, recombinases, and methylases.
• ZFs zinc finger proteins
• TALEs transcription activator- like effectors
• DNA binding domains such as a receptor- ligand pair
• ZF or TALE protein the receptor
• DNA ligand
• a total of 16 zinc finger proteins were designed and generated, providing concrete examples of systematically programmed polynucleotide-binding domain/polynucleotide pairs.
• the protein sequences and target dsDNA sequences are provided in Fig. 1.
• each domain's N terminus to an Ig ⁇ -chain leader sequence and each C-terminus to the platelet derived growth factor (PDGF) transmembrane domain.
• PDGF platelet derived growth factor
• sZFs Two aspects of this sZF-DNA interaction were of note: first, sZFs were observed to bind to both single and double stranded DNA molecules (Fig. 4a); however, the former interaction was abrogated in the presence of competitor dsDNA (here Salmon Sperm DNA). Second, sZFs also non- specifically bound to most dsDNA, but again in the presence of competitor dsDNA, binding to only their cognate target dsDNA was retained (Fig. 4b). Similar results were obtained using FACS based assays (Fig. 4c). Thus fusion gene-expressing cells specifically bind target dsDNA probes (Fig. 3).
• Example 3 Verification of predicted polynucleotide -binding domain/polynucleotide pairs in cells To verify the 16 zinc finger proteins and their dsDNA target sequences would interact as predicted, and further that they would do so when the polynucleotide-binding domain was expressed at the cell surface, the zinc fingers were modified for expression in cells, expressed in cells, and tested for binding of complementary dsDNA probes as described in Example 2 (Fig. 5a-c).
• sZFs were observed to have different binding affinities for their target dsDNA (Fig. 6a). Specifically, as assayed by both fluorescence intensity and duration of binding, some sZFs bound their targets strongly (ZFs 1, 3, 8, 12, 13, 15, 16), some moderately strongly (ZFs 2, 4, 5, 6, 7, 10, 14), and others only weakly (ZFs 9, 1 1).
• Weak binders required low concentrations of dsDNA probes in the solution to prevent loss of fluorescence over time.
• cells expressing moderate to strong binders show little loss of fluorescence intensity over long durations of time, a feature that greatly facilitates ease of imaging.
• each sZF for its ability to bind its own target dsDNA sequence and also target dsDNA sequences corresponding to the other zinc finger proteins, i.e., a total of 16x16 interactions were probed to generate a complete cross reactivity profile (Fig. 6b).
• ZFs 1, 8, 13 The strong ZF binders were particularly susceptible to this phenomenon.
• almost all the zinc fingers were observed to bind the ZF16 target dsDNA, likely in part to the high poly-G rich content of this sequence.
• sZFs are to serve as faithful barcodes they must also enable differential labeling of cells in complex mixtures.
• the ability to re-probe a cell with different labels or functional tags in a sequential manner is also desired.
• the zinc-finger dsDNA interaction is a non-covalent interaction it should be feasible to displace the latter using a competing dsDNA ligand.
• sZFs we examined the dsDNA dissociation kinetics from sZFs. In general assayed sZFs were observed to have a high affinity for their target dsDNA thus demonstrating low rates of dissociation that enabled long-term visualization of the tagged cells.
• each sZF has a corresponding probe comprising two parts: a dsDNA portion that specifically binds the zinc finger protein, and a single-stranded portion that is designed to include several hybridization sites (Fig. 15a-b).
• hybridization sites provide a unique sequence code for the sZF, which is decoded by probing the sites sequentially as follows: in step 1, a fluorophore tagged complementary oligonucleotide is hybridized to its target site enabling a first fluorescence readout; in step 2, two adj acent complementary oligonucleotides are annealed, the first bearing a quencher that suppresses the step 1 fluorescence signal, and the second bearing another fluorophore that enables a second fluorescent readout and so on.
• each sZF is encoded by a sequence of fluorescent states that are progressively read by turning each state on and then off.
• Example 6 Use of zinc finger binding domains as reporters of endogenous cellular activity
• sZFs could also serve as surrogate reporters of endogenous cellular activity.
• lentiviral vectors with small molecule (tetracycline and cumate) inducible promoters to drive sZF expression were constructed. Stable transductions of 293T and HeLa cells were performed, and upon small molecule induction sZF expression could be readily detected by the ability of the cells to bind dsDNA molecules (Fig. 16). Expression of sZFs from the tet responsive promoters was observed to be higher than from the cumate inducible promoters (Fig. 17), but both inducible systems showed robust induction and can be used as versatile tools for barcoding cells. Together we conclude that like fluorescent proteins, sZFs in addition to labeling cells can also serve as reporters of transcriptional activity in multiple cell types.

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