WO2022159193A1 - Proximity-dependent photoactivation - Google Patents

Proximity-dependent photoactivation Download PDF

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Publication number
WO2022159193A1
WO2022159193A1 PCT/US2021/062616 US2021062616W WO2022159193A1 WO 2022159193 A1 WO2022159193 A1 WO 2022159193A1 US 2021062616 W US2021062616 W US 2021062616W WO 2022159193 A1 WO2022159193 A1 WO 2022159193A1
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molecule
polypeptide
rhodamine dye
fluorophore
effector
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PCT/US2021/062616
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French (fr)
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WO2022159193A9 (en
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Thomas Graham
Robert Tjian
Xavier Darzacq
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The Regents Of The University Of California
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

Definitions

  • a number of methods for detecting interaction of two molecules are available. Such methods include, e.g., surface plasmon resonance, fluorescence resonance energy transfer, bioluminescence resonance energy transfer, and bimolecular fluorescence complementation.
  • surface plasmon resonance fluorescence resonance energy transfer
  • bioluminescence resonance energy transfer bioluminescence resonance energy transfer
  • bimolecular fluorescence complementation bimolecular fluorescence complementation
  • the present disclosure provides methods of detecting proximity of a first molecule to a second molecule, where the first molecule comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore in a proximity-dependent manner; and where the second molecule comprises the effector fluorophore.
  • the present disclosure also provides systems for carrying out a method of the present disclosure.
  • FIG. 1 provides a schematic depiction of an embodiment of the present disclosure.
  • FIG. 2A-2B depict reactivation of Janelia Fluor X 650 (JFX650), dependent on colabeling a pentameric HaloTag fusion protein with Janelia Fluor 549 (JF549).
  • FIG. 3A-3B depict alternating proximity-dependent and proximity-independent reactivation of Janelia Fluor 646 (JF646).
  • FIG. 4 depicts proximity-dependent reactivation of JF646 in fixed cells.
  • FIG. 5 depicts reactivation of JFX650 when a HaloTag-SNAP fusion protein is doublelabeled with JFX650 SNAP ligand and JF549 HaloTag ligand.
  • FIG. 6A-6C depict multiple cycles of alternating illumination.
  • FIG. 7 depicts data showing that reactivation of JFX650 by JF549 + 561 nm light depends on proximity of the two dyes within the same molecular complex.
  • FIG. 8 depicts detection of inducible dimerization using proximity-dependent photoactivation.
  • FIG. 9 depicts detection of androgen receptor (AR) self-association using proximitydependent photoactivation.
  • FIG. 10 depicts a single -molecule displacement histogram from single-particle tracking (SPT) combined with proximity-dependent photoactivation.
  • SPT single-particle tracking
  • FIG. 11A-11B depict selective highlighting of double-labeled molecules using proximity-dependent reactivation.
  • FIG. 12 depicts the effect of a 67-amino acid rigid alpha helical spacer on 561 nm photoactivation.
  • FIG. 13A-13C depict proximity-assisted photoactivation (PAPA) of JFX650 by JF549.
  • FIG. 14A-14D depict properties of JFX650 reactivation.
  • FIG. 15A-15E depict PAPA between various sender-receiver pairs.
  • FIG. 16A-16B depict SDS-PAGE analysis of linker constructs.
  • FIG. 17A-17B depict a comparison of distance-dependence of PAPA and FRET
  • FIG. 18A-18C depict detection of inducible dimerization using PAPA.
  • FIG. 19A-19B depict SDS-PAGE and PAPA traces of FRB-FKBP.
  • FIG. 20A-20F depict an example of “unmixing” of defined 2-component mixtures using
  • FIG. 21A-21B depict SDS-PAGE gels of defined 2-component mixtures and 1- component controls.
  • FIG. 22A-22H depict additional analyses of PAPA-fastSPT experiment with 2- component controls.
  • FIG. 23A-23I depict PAPA-SPT analysis of single-component controls.
  • FIG. 24A-24C depict PAPA analysis of androgen receptor.
  • FIG. 25A-25F depict another example of “unmixing” of defined 2-component mixtures using PAPA.
  • FIG. 26A-26D depict analysis of mammalian androgen receptor using PAPA-SPT.
  • FIG. 27 schematically depicts use of PAPA to spotlight protein-protein interactions.
  • peptide refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • the terms also include polypeptides that have co- translational (e.g., signal peptide cleavage) and post- translational modifications of the polypeptide, such as, for example, disulfide-bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage, and the like.
  • polypeptide refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art) to the native sequence, as long as the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts that produce the proteins, or errors due to polymerase chain reaction (PCR) amplification or other recombinant DNA methods.
  • modifications such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art) to the native sequence, as long as the protein maintains the desired activity.
  • modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts that produce the proteins, or errors due to polymerase chain reaction (PCR) amplification or other recombinant DNA methods.
  • polynucleotide oligonucleotide
  • nucleic acid e.g., deoxyribonucleotides, or a combination thereof
  • polynucleotide includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • polynucleotide and “nucleic acid” should be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
  • a polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST. See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10.
  • FASTA is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc.
  • GCG Genetics Computing Group
  • Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA.
  • alignment programs that permit gaps in the sequence.
  • the Smith- Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997).
  • the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).
  • heterologous used in reference to nucleic acids or polypeptides, means that these molecules are not naturally occurring in the environment (e.g., a cell) from which the heterologous nucleic acid or polypeptide was derived.
  • a fusion polypeptide can comprise a target polypeptide and a heterologous fusion partner, where the heterologous fusion partner does not normally exist in a single polypeptide chain in nature with the target polypeptide.
  • linking or "linker” as in “linking group,” “linker moiety,” etc., is meant a linking moiety that connects two groups (e.g., a rhodamine dye to a first molecule; a rhodamine dye to an affinity agent; an effector fluorophore to a second molecule; an effector fluorophore to an affinity agent; and the like) via covalent bonds.
  • the linker may be linear, branched, cyclic or a single atom.
  • linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and linking moieties containing functional groups including, without limitation: amido (-NH-CO-), ureylene (-NH-CO-NH-), imide (-CO-NH-CO-) , epoxy (-O-), epithio (-S-), epidioxy (-O-O-), epidithio (-S-S-), carbonyldioxy (-O-CO-O-), alkyldioxy (-O- (CH2)n-O-), epoxyimino (-0-NH-), epimino (-NH-), carbonyl (-CO-), etc.
  • one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom.
  • the bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone.
  • the linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group.
  • a linker may include, without limitations, poly(ethylene glycol) unit(s) (e.g., -(CH2-CH2-O)-); amino acids; ethers; thioethers; amines; alkyls (e.g., (Ci-Ci2)alkyl), which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1- methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1 -dimethylethyl (t-butyl), and the like.
  • poly(ethylene glycol) unit(s) e.g., -(CH2-CH2-O)-
  • amino acids ethers; thioethers; amines; alkyls (e.g., (Ci-Ci2)alkyl)
  • alkyls e.g., (Ci-Ci2)alkyl
  • alkyls e
  • the linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3, or 4 atoms, of the cyclic group are included in the backbone.
  • a linker may be cleavable or non-cleavable. Any convenient orientation and/or connections of the linkers to the linked groups may be used.
  • Other examples of linking groups include oligonucleotides and polypeptides.
  • binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
  • a “host cell,” as used herein, denotes an in vivo or in vitro eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which eukaryotic or prokaryotic cells can be, or have been, used as recipients for a nucleic acid (e.g., an expression vector), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
  • a “recombinant host cell” (also referred to as a “genetically modified host cell”) is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector.
  • a genetically modified prokaryotic host cell e.g., a bacterium
  • a heterologous nucleic acid e.g., an exogenous nucleic acid that is foreign to (not normally found in nature in) the prokaryotic host cell, or a recombinant nucleic acid that is not normally found in the prokaryotic host cell.
  • a genetically modified eukaryotic host cell is genetically by virtue of introduction into a suitable eukaryotic host cell of a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in the eukaryotic host cell.
  • a heterologous nucleic acid e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in the eukaryotic host cell.
  • genetic modification refers to a permanent or transient genetic change induced in a cell following introduction of new nucleic acid (e.g., DNA exogenous to the cell) into the cell.
  • Genetic change (“modification”) can be accomplished either by incorporation of the new nucleic acid into the genome of the host cell, or by transient or stable maintenance of the new nucleic acid as an episomal element.
  • a permanent genetic change is generally achieved by introduction of new DNA into the genome of the cell. Permanent changes can be introduced via extrachromosomal elements such as plasmids and expression vectors, which may contain one or more selectable markers to aid in their maintenance in the recombinant host cell.
  • Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like.
  • the choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (i.e. in vitro, ex vivo, or in vivo).
  • a general discussion of these methods can be found in Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.
  • the present disclosure provides methods of detecting proximity of a first molecule to a second molecule, where the first molecule comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore in a proximity-dependent manner; and where the second molecule comprises the effector fluorophore.
  • the present disclosure also provides systems for carrying out a method of the present disclosure.
  • the present disclosure provides proximity-dependent photoactivation methods for detecting proximity of a first molecule to a second molecule, where the first molecule comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore in a proximity-dependent manner; and where the second molecule comprises the effector fluorophore.
  • the term “effector fluorophore” is used interchangeably herein with “sender fluorophore.”
  • the “effector fluorophore” may also include weakly fluorescent chromophores that are not typically used for fluorescence imaging.
  • FIG. 1 provides a schematic depiction of an embodiment of the present disclosure.
  • a sample comprises a first molecule (“X”) that is labeled with a “receiver” fluorophore (e.g., Janelia Fluor 646 (JF646)) and a second molecule (“Y”) that is labeled with a “sender” fluorophore (e.g., Janelia Fluor 549 (JF549)) (left panel).
  • the sample is first irradiated with light of a first wavelength (e.g., 633 nm) that inactivates JF646 and converts it to a dark state (middle panel).
  • a first wavelength e.g., 633 nm
  • the sample is then irradiated with light of a second wavelength (e.g., 561 nm), thereby exciting the JF549.
  • excitation of JF549 causes reactivation of the adjacent darkstate JF646 fluorophore.
  • Proximity of molecule X and molecule Y is thus determined by detecting fluorescence from JF646 upon subsequent direct excitation (e.g., with 633 nm light).
  • JF646 is reactivated following activation of JF549 only when molecule X and molecule Y are in sufficient proximity to one another.
  • the present disclosure provides a method of detecting proximity of a first molecule to a second molecule, the method comprising: a) contacting the first molecule with the second molecule to form a contacted sample, where: i) the first molecule comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore in a proximity-dependent manner; and ii) the second molecule comprises the effector fluorophore, where the contacting is carried out under conditions that inactivate the rhodamine dye; b) exciting the effector fluorophore with a second wavelength of light; and c) detecting fluorescence from the rhodamine dye in the contacted sample following excitation of the effector fluorophore.
  • Excitation of the effector fluorophore with the second wavelength of light results in reactivation of the rhodamine dye.
  • Fluorescence from the rhodamine dye indicates that the first molecule and the second molecule are in proximity to one another in the contacted sample.
  • fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule were in proximity to one another in the contacted sample at the time the effector fluorophore was excited. In other words, sustained contact between the first and the second molecule is not required in order for the rhodamine dye to remain reactivated.
  • reactivation requires only that the first and second molecules be in contact at some time when the effector fluorophore is excited.
  • fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule were in proximity to one another in the contacted sample at the time the effector fluorophore was excited, even if fluorescence from the reactivated rhodamine dye is detected (and/or measured) at a time point after the effector fluorophore is excited.
  • fluorescence from the reactivated rhodamine dye can be measured at a time point that is from about 1 second (or less than 1 second) to about 1 hour (or more than 1 hour) after the effector fluorophore is excited.
  • fluorescence from the reactivated rhodamine dye can be measured at a time point that is from about 1 second to about 5 seconds, from about 5 seconds to about 10 seconds, from about 10 seconds to about 15 seconds, from about 15 seconds to about 30 seconds, from about 30 seconds to about 1 minute, from about 1 minute to about 5 minutes, from about 5 minutes to about 10 minutes, from about 10 minutes to about 15 minutes, from about 15 minutes to about 30 minutes, or from about 30 minutes to about 1 hours, or more than 1 hour, after the effector fluorophore is excited.
  • fluorescence from the reactivated rhodamine dye is measured within about 10 seconds of the time that the effector fluorophore is excited.
  • fluorescence from the reactivated rhodamine dye is measured at a time point that is from about 1 millisecond to about 5 milliseconds, from about 5 milliseconds to about 10 milliseconds, from about 10 milliseconds to about 15 milliseconds, from about 15 milliseconds to about 100 milliseconds, from about 100 milliseconds to about 250 milliseconds, from about 250 milliseconds to about 500 milliseconds, or from about 500 milliseconds to about 1 second, after the effector fluorophore is excited.
  • detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are within 25 nm of one another. In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are within 20 nm of one another. In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are within 15 nm of one another. In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are within 10 nm of one another.
  • detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 0.1 nm to about 25 nm from one another; e.g., detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 0.1 nm to about 0.5 nm, from about 0.5 nm to about 1 nm, from about 1 nm to about 2 nm, from about 2 nm to about
  • detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 0.1 nm to about 1 nm from one another. In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 0.1 nm to about 5 nm from one another.
  • detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 0.1 nm to about 10 nm from one another. In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 0.5 nm to about 5 nm from one another. In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 1 nm to about 5 nm from one another.
  • detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 1 nm to about 10 nm from one another In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are less than about 10 nm apart from one another; e.g., less than about 10 nm, less than about 7 nm, less than about 5 nm, less than about 3 nm, or less than about 1 nm apart from one another.
  • Proximity of a first molecule and a second molecule to one another can comprise binding of the first molecule to the second molecule.
  • “proximity” of a first molecule and a second molecule to one another is due to non-covalent interaction of the first molecule with the second molecule, where non-covalent interactions can include ionic interactions, electrostatic interactions, van der Waals interactions, and the like.
  • “proximity” of a first molecule and a second molecule to one another is due to covalent binding of the first molecule to the second molecule.
  • the proximity of the first molecule to the second molecule occurs for a period of time of less than 1 minute.
  • the proximity of the first molecule to the second molecule occurs for a period of time of less than about 60 seconds, less than about 45 seconds, less than about 30 seconds, less than about 15 seconds, less than about 10 seconds, or less than about 5 seconds.
  • the proximity of the first molecule to the second molecule occurs for a period of time of from about 1 second to about 60 seconds; e.g., from about 1 second to about 5 seconds, from about 5 seconds to about 10 seconds, from about 10 seconds to about 15 seconds, from about 15 seconds to about 30 seconds, from about 30 seconds to about 45 seconds, or from about 45 seconds to about 60 seconds.
  • the proximity of the first molecule to the second molecule occurs for a period of time of less than 1 second.
  • the proximity of the first molecule to the second molecule occurs for a period of time of less than about 1 second, less than about 500 milliseconds (msec), less than about 250 msec, less than about 100 msec, less than about 50 msec, less than about 25 msec, less than about 10 msec, less than about 5 msec, or less than about 1 msec.
  • the proximity of the first molecule to the second molecule occurs for a period of time of from about 1 msec to about 1 second; e.g., the proximity of the first molecule to the second molecule occurs for a period of time of from about 1 msec to about 10 msec, from about 10 msec to about 25 msec, from about 25 msec to about 50 msec, from about 50 msec to about 100 msec, from about 100 msec to about 250 msec, from about 250 msec to about 500 msec, or from about 500 msec to 1 second.
  • the proximity of the first molecule to the second molecule occurs for a period of time of less than 1 millisecond; for example, in some cases, the proximity of the first molecule to the second molecule occurs for a period of time of less than 1 millisecond, less than 500 nanoseconds, less than 250 nanoseconds, less than 100 nanoseconds, less than 50 nanoseconds, less than 25 nanoseconds, less than 10 nanoseconds, less than 5 nanoseconds, or about 1 nanosecond. In some cases, the proximity of the first molecule to the second molecule occurs for a period of time of from about 1 nanosecond to about 1 millisecond.
  • the proximity of the first molecule to the second molecule occurs for a period of time of from about 1 nanosecond to about 5 nanoseconds, from about 5 nanoseconds to about 10 nanoseconds, from about 10 nanoseconds to about 25 nanoseconds, from about 25 nanoseconds to about 50 nanoseconds, from about 50 nanoseconds to about 100 nanoseconds, from about 100 nanoseconds to about 250 nanoseconds, from about 250 nanoseconds to about 500 nanoseconds, or from about 500 nanoseconds to about 1 millisecond.
  • the proximity of the first molecule to the second molecule occurs for a period of time of more than 1 minute.
  • the proximity of the first molecule to the second molecule occurs for a period of time of from about 1 minute to about 10 minutes, or more than 10 minutes.
  • a method of the present disclosure provides for detection of proximity of a first molecule to a second molecule, where the first molecule comprises a rhodamine dye, and the second molecule comprises an effector fluorophore.
  • Rhodamine dyes are known in the art, and any rhodamine dye can be suitable for use in a method of the present disclosure.
  • a suitable rhodamine dye is a silicon- rhodamine dye.
  • a suitable rhodamine dye is a Janelia-Fluor, silicon-rhodamine (Si-rhodamine)-containing dye. See, e.g., Grimm et al. (2017) ACS Central Science 3:975; Grimm et al. (2015) Nature Methods 12:244; and WO 2017/201531.
  • a suitable rhodamine dye is JF646 (Compound 26 of Grimm et al. (2015) Nature Methods 12:244). JF646 has the following structure:
  • JF635 has the following structure:
  • suitable rhodamine dyes are Janelia Fluor X 646 (JFX646) or Janelia Fluor X 650 (JFX650).
  • HaloTag ligand conjugates of JFX646 and JFX650 have the following structures (where D is deuterium):
  • a suitable rhodamine dye is Janelia Fluor 585 (JF585) (Zheng et al., ACS
  • JF585 has the following structure:
  • JF608 a suitable rhodamine dye is Janelia Fluor 608 (JF608) (Zheng et al., ACS Cent. Sci. 2019, DOI: 10.1021/acscentsci.9b00676). JF608 has the following structure:
  • Alexa Fluor 633 has the following structure: Effector fluorophores
  • Fluorophores e.g., fluorescent dyes; fluorescent proteins
  • fluorescent dyes are molecules (e.g., dyes; polypeptides) that, when irradiated with light of a wavelength (a first wavelength) which they absorb, can emit light of a different wavelength (a second wavelength).
  • Suitable effector fluorophores include, e.g., a rhodamine dye; a fluorescent polypeptide; and the like.
  • effector fluorophore is used interchangeably herein with “sender fluorophore.”
  • a suitable effector fluorophore is one that reactivates the inactivated rhodamine dye (that is linked to the first molecule) in a proximitydependent manner; i.e., the effector fluorophore is linked to the second molecule (directly or via a linker) and reactivates that inactivated rhodamine dye (that is linked to the first molecule) only when the first molecule and the second molecule are in sufficiently close proximity to one another (as described above).
  • Suitable effector fluorophores include, but are not limited to, JF549, Alexa Fluor 532, Alexa Fluor 549, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, tetramethylrhodamine, ATTO 532, ATTO Rho6G, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol l, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO590, ATTO Rhol3, or ATTO594.
  • JF526 Janelia fluorophore 526
  • the effector fluorophore is a fluorescent polypeptide such as mKO, mK02, DsRed, Turbo red fluorescent protein (RFP), TagRFP, tdTomato, mOrange, or O- GECO1.
  • a DsRed polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
  • an mK02 polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
  • JF549 has the following structure:
  • JFX549 The structure of JFX549 is as follows:
  • the effector fluorophore is not a cyanine dye such as Cy3 or Cy5.
  • the first molecule and the second molecule can be any of a variety of molecules, including synthetic (non-naturally-occurring) molecules, biomolecules, nucleotides, nucleotide analogs, sugars, sugar analogs, small molecules (e.g., small molecule drugs), and the like.
  • Suitable molecules include, but are not limited to, polypeptides, nucleic acids, lipids, sugars (e.g., mannose, lactose, glucose, galactose, N-acetyl-glucosamine, N-acetyl-galactosamine, fucose, and the like), polysaccharides, and molecules comprising one or more of the foregoing.
  • a molecule can be a lipopolysaccharide, a glycoprotein, a glycolipid, a lipoprotein, a proteoglycan, and the like.
  • Suitable biomolecules include, e.g., RNA (e.g., mRNA, gRNA, miRNA, piRNA, sgRNA, shRNA, siRNA, stRNA, sncRNA, tncRNA, snoRNA, smRNA, shRNA, snRNA, tRNA, tracrRNA, or other small non-coding RNA), DNA, peptide nucleic acids, combinations of RNA and DNA (e.g., chimeraplasts), and the like.
  • RNA e.g., mRNA, gRNA, miRNA, piRNA, sgRNA, shRNA, siRNA, stRNA, sncRNA, tncRNA, snoRNA, smRNA, shRNA, snRNA, tRNA,
  • the first molecule and the second molecule are in vitro outside of a cell (e.g., in an acellular environment). In some cases, the first molecule and the second molecule are present in a living cell in vitro. In some cases, the first molecule and the second molecule are present in a multicellular organism; e.g., in some cases, the first molecule and the second molecule are in vivo. Where the first molecule and the second molecule are in vivo, in some cases, the first molecule and the second molecule are inside of a cell.
  • the first molecule and the second molecule are in vivo, in some cases, the first molecule and the second molecule are outside of a cell; e.g., the first molecule and the second molecule are in extracellular fluid. In some cases, the first molecule is present on the surface of a cell; and the second molecule is outside of the cell. In some cases, the first molecule and the second molecule are inside a living cell within a cultured organoid or tissue explant. In some cases, the first molecule and the second molecule are in the same organelle within a eukaryotic cell. In some cases, the first molecule and the second molecule are in different organelles within a eukaryotic cell.
  • Suitable polypeptide biomolecules include, e.g., receptors, immunomodulatory polypeptides, cytokines, chemokines, peptide hormones, differentiation factors, growth factors, enzymes, antibodies, peptide neurotransmitters, transcriptional repressors, transcriptional activators, nucleic acid binding polypeptides (e.g., chromatin and the like), structural polypeptides, and the like.
  • Suitable polypeptide biomolecules include structural proteins; receptors; enzymes; cell surface proteins; proteins integral to the function of a cell; proteins involved in catalytic activity; proteins involved in motor activity; proteins involved in helicase activity; proteins involved in metabolic processes (anabolism and catabolism); proteins involved in antioxidant activity; proteins involved in proteolysis; proteins involved in biosynthesis; proteins having kinase activity; proteins having oxidoreductase activity; proteins having transferase activity; proteins having hydrolase activity; proteins having lyase activity; proteins having isomerase activity; proteins having ligase activity; proteins having enzyme regulator activity; proteins having signal transducer activity; structural polypeptides; polypeptides having binding activity; receptor polypeptides; proteins involved in cell motility; proteins involved in membrane fusion; proteins involved in cell communication; proteins involved in regulation of biological processes; proteins involved in development; proteins involved in cell differentiation; proteins involved in response to stimulus; behavioral proteins; cell adhesion proteins; proteins involved in cell death; proteins involved in transport (including protein transporter activity, nuclear transport
  • Suitable polypeptides can be of any of a variety of origins, and can thus include mammalian polypeptides, insect polypeptides, arachnid polypeptides, invertebrate polypeptides, viral polypeptides, bacterial polypeptides, archaeal polypeptides, and the like.
  • Suitable biomolecules include nucleic acids.
  • the nucleic acid is a DNA molecule.
  • the nucleic acid is an RNA molecule.
  • the nucleic acid comprises both deoxyribonucleotides and ribonucleotides.
  • the nucleic acid is a single-stranded DNA molecule.
  • the nucleic acid is a double-stranded DNA molecule.
  • the nucleic acid is a single-stranded RNA molecule.
  • Suitable nucleic acids include, e.g., a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a ribozyme, and the like.
  • Suitable nucleic acids include nucleic acids that are or act as siRNAs or other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, self-cleaving RNAs, ribozymes, fragment thereof and/or variants thereof (such as Peptidyl transferase 23S rRNA, RNase P, Group I and Group II introns, GIRI branching ribozymes, Leadzyme, Hairpin ribozymes, Hammerhead ribozymes, HDV ribozymes, Mammalian CPEB3 ribozyme, VS ribozymes, glmS ribozymes, CoTC ribozyme, etc.), micro
  • Suitable molecules include small molecules such as drugs, e.g., where a small molecule can have a molecular weight in the range of from about 5 Daltons to 2500 Daltons. Small molecules may comprise functional groups necessary for structural interaction with other molecules such as proteins, where such interactions may be via hydrogen bonding. Small molecules can include one or more of an amine, a carbonyl, a hydroxyl, or a carboxyl group. Small molecules may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Small molecule can also include amino acids, sugars, oligosaccharides, fatty acids, steroids, purines, or pyrimidines, as well as derivatives, structural analogs or combinations thereof.
  • Suitable small molecules include, e.g., vitamins, neurotransmitters, ligands for receptors, monosaccharides, disaccharides, and the like.
  • Suitable small molecules include therapeutic agents, e.g., cancer chemotherapeutic agents, anti-inflammatory agents, anti-hypertensive agents, and the like.
  • the first molecule or the second molecule is a synthetic polymer.
  • Suitable synthetic polymers include, but are not limited to, polyalkylenes such as polyethylene and polypropylene and polyethyleneglycol (PEG); poly chloroprene; polyvinyl ethers such as poly(vinyl acetate); polyvinyl halides such as poly (vinyl chloride); poly siloxanes; polystyrenes; polyurethanes; polyacrylates such as poly(methyl (meth)acrylate), poly(ethyl (meth) acrylate), poly(n-butyl(meth)acrylate), poly(isobutyl (meth) acrylate), poly(tert-butyl (meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly
  • the first molecule comprises a first polypeptide; and the second molecule comprises a second polypeptide.
  • the first molecule comprises a nucleic acid; and the second molecule comprises a second nucleic acid.
  • the first molecule comprises a nucleic acid; and the second molecule comprises a polypeptide.
  • the first molecule comprises a polypeptide; and the second molecule comprises a nucleic acid.
  • the first molecule comprises a lipid; and the second molecule comprises a polypeptide.
  • the first molecule comprises a polypeptide; and the second molecule comprises a lipid.
  • the first molecule comprises a polysaccharide; and the second molecule comprises a polypeptide.
  • the first molecule comprises a polypeptide; and the second molecule comprises a polysaccharide.
  • the first molecule and/or the second molecule can include a heterologous moiety such as an affinity tag, a self-labelling polypeptide, and the like.
  • the first molecule and/or the second molecule can include, as a heterologous moiety, an affinity tag.
  • Suitable affinity tags include, for example, a histidine tag (His tag), a chitin-binding domain, a calmodulin tag, a polyglutamate tag, a maltose binding protein, glutathione-S- transferase, an S-tag, an SBP-tag (Keefe et al. (2001) Protein Expr. Purif.
  • Strep-tag e.g., an 8-amino acid peptide of the amino acid sequence Trp-Ser-His-Pro-Gln-Phe- Glu-Lys
  • fluorescent protein e.g., an 8-amino acid peptide of the amino acid sequence Trp-Ser-His-Pro-Gln-Phe- Glu-Lys
  • a fluorescent protein e.g., an 8-amino acid peptide of the amino acid sequence Trp-Ser-His-Pro-Gln-Phe- Glu-Lys
  • a fluorescent protein e.g., an 8-amino acid peptide of the amino acid sequence Trp-Ser-His-Pro-Gln-Phe- Glu-Lys
  • a fluorescent protein e.g., an 8-amino acid peptide of the amino acid sequence Trp-Ser-His-Pro-Gln-Phe- Glu-Lys
  • SpyCatcher e.g., a peptide of the sequence: AHIVMDAYKPTK (SEQ ID NO:3)
  • SnoopTag e.g., a peptide of the sequence: KLGDIEFIKVNK (SEQ ID NO:4)
  • the first molecule and/or the second molecule can include, as a heterologous moiety, a self-labelling polypeptide.
  • Suitable self-labelling polypeptides include a SNAP polypeptide, a CLIP polypeptide, or a HALO polypeptide.
  • a halo-based oligonucleotide binder (HOB) polypeptide See, e.g., Kossman et al. (2016) Chembiochem. 17:1102. A HOB polypeptide binds chlorohexyl moieties.
  • trimethoprim an engineered form of E. coli dihydrofolate reductase (DHFR) that forms a non-covalent high-affinity complex with trimethoprim derivatives.
  • TMP trimethoprim
  • DHFR E. coli dihydrofolate reductase
  • a SNAP polypeptide can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEVPAPAAVLGGPEPL MQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYSHLA ALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLAVKEWLLAHEGHRL GKPGLG (SEQ ID NO:5).
  • a SNAP polypeptide binds O 6 -benzylguanine (BG).
  • a CLIP polypeptide can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEVPAPAAVLGGPEPL IQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISESHLA ALVGNPAATAAVNTALDGNPVPILIPCHRVVQGDSDVGPYLGGLAVKEWLLAHEGHRL GKPGLG (SEQ ID NO:6).
  • a CLIP polypeptide can bind O 2 -benzylcytosine (BC).
  • a HALO polypeptide can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTH RCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAK RNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVV RPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKL LFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEI
  • the first molecule and/or the second molecule can include, as a heterologous moiety, a sortase.
  • Suitable sortases include sortase A (SrtA), sortase B (SrtB), sortase C (SrtC), sortase D (SrtD), sortase E (SrtE), sortase F (SrtF), and variants thereof.
  • a method of the present disclosure provides for detection of proximity of a first molecule to a second molecule, where the first molecule comprises a rhodamine dye, and the second molecule comprises an effector fluorophore.
  • the rhodamine dye can be linked to the first molecule directly or via a linker.
  • effector fluorophore can be linked to the second molecule directly or via a linker.
  • a rhodamine dye is linked to the first molecule, or an effector fluorophore is linked to the second molecule, via a HALO tag, a SNAP tag, a CLIP tag, a sortase polypeptide, dihydrofolate reductase (DHFR), tetracysteine, and the like.
  • rhodamine dye or the effector fluorophore is linked to an affinity agent such as a benzylnucleoside base or a chloropyrimidine.
  • rhodamine dye or the effector fluorophore is linked to a benzylnucleoside base, such as benzylguanine or benzylcytosine. In some cases, the rhodamine dye or the effector fluorophore is linked to benzylguanine. In embodiments where the rhodamine dye or the effector fluorophore is linked to benzylguanine, the benzylguanine can provide for covalent binding to a SNAP tag, e.g., where the first molecule or the second molecule comprises the SNAP tag.
  • the rhodamine dye or the effector fluorophore is linked to benzylcytosine.
  • the benzylcytosine can provide for covalent binding to a CLIP tag, e.g., where the first molecule or the second molecule comprises the CLIP tag.
  • the affinity agent is a chloropyrimidine; a chloropyrimidine can bind to a SNAP tag.
  • the affinity agent is O 6 -benzylguanine (e.g., a substrate for SNAP).
  • the affinity agent is O 6 -(5-pyridylmethyl)guanine (e.g. a substrate for SNAP).
  • Suitable affinity agents also include alkyl derivatives, such as haloalkyl derivatives where one or more hydrogen atoms in an alkyl or alkyl derivative is replaced by a halogen, e.g., fluoro, chloro, or bromo.
  • the haloalkyl derivative is a fluoroalkane.
  • the haloalkyl derivative is a chloroalkane.
  • the haloalkyl derivative is a bromoalkane.
  • the affinity agent is chloroalkane, such as CKCEL OCELCEL ⁇ .
  • the affinity agent is chloroalkane
  • the chloroalkane affinity agent can provide for covalent binding to a HALO tag.
  • a rhodamine dye is attached to the first molecule and/or the effector fluorophore is attached to the second molecule via a linker.
  • a linker is any suitable moiety capable of linking, connecting, or tethering a rhodamine dye to a first molecule or an effector fluorophore to a second molecule.
  • a linker can be a polymer of one or more repeating or nonrepeating monomer units (e.g., nucleic acid, amino acid, carbon-containing polymer, carbon chain, etc.). In some cases, a linker is a peptide linker.
  • a linker can comprise any chemical moiety with a functional (or reactive) group that can be linked to a rhodamine dye (or a moiety linked to a rhodamine dye) or an effector fluorophore (or a moiety linked to an effector fluorophore). Any suitable moiety capable of tethering the signal and interaction elements may find use as a linker.
  • linker groups can be used. Suitable linkers can comprise, e.g., an alkyl group, a methylene carbon chain, an ether, a polyether, an alkyl amide linker, a peptide linker, a modified peptide linker, a poly(ethylene glycol) (PEG) linker, a streptavidin-biotin or avidinbiotin linker, polyaminoacids (e.g.
  • polylysine functionalized PEG, polysaccharides, glycosaminoglycans, dendritic polymers an oligonucleotide linker, a phospholipid linker, an alkenyl chain, an alkynyl chain, a disulfide, or a combination thereof.
  • the linker is cleavable.
  • Peptide linkers include, e.g., peptides from about 4 amino acids to about 300 amino acids in length.
  • suitable peptide linkers can be from about 4 amino acids to about 25 amino acids in length, from about 25 amino acids to about 50 amino acids in length, from about 50 amino acids to about 100 amino acids in length, from about 100 amino acids to about 200 amino acids in length, or from about 200 amino acids to about 300 amino acids in length.
  • Suitable peptide linkers include glycine-serine polymers such as (GGGGS)n (SEQ ID NO:8), (GSGGS)n (SEQ ID NO:9), (GGGS)n (SEQ ID NO: 10), and the like, where n is an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • Suitable peptide linkers include, e.g., (GGGGS)n (SEQ ID NO:8), where n is 1, 2, or 3.
  • Suitable peptide linkers also include longer (up to about 300 amino acids) artificial low-complexity sequences that are designed to be flexible, non-self- interacting, and solvent-exposed.
  • Suitable peptide linkers include flexible polypeptide sequences from naturally occurring proteins.
  • the conformational flexibility of the linker(s) will allow the rhodamine dye and the effector fluorophore to come in contact repeatedly during a photoactivation pulse. Varying the linker length and activation pulse duration can also provide a means of gauging the spatial distance between the first molecule and the second molecule, and of extending the distance range of detection.
  • Suitable linkers include peptide linkers (e.g., flexible peptide linkers), oligonucleotides, and synthetic polymers.
  • a rhodamine dye can be linked to a first molecule using any of a variety of standard chemistries. Suitable chemistries include, e.g., maleimide/thiol; thiol/thiol; pyridyldithiol/thiol; succinimidyl iodoacetate/thiol; N-succinimidylester (NHS ester), sulfodicholorphenol ester (SDP ester), or pentafluorophenyl-ester (PFP ester)/amine; bissuccinimidylester/amine; imidoesters/amines; hydrazine or amine/aldehyde, dialdehyde or benzaldehyde; isocyanate/hydroxyl or amine; carbohydrate-periodate/hy dr azine or amine; diazirine/aryl azide chemistry; pyridyld
  • Suitable chemistries also include click chemistry.
  • an effector fluorophore can be linked to a second molecule using any of a variety of standard chemistries, including any of the aforementioned chemistries.
  • Proteins may be labeled with rhodamine dyes or effector dyes by incorporation of unnatural amino acids (reviewed by Elia et al., 2020, The FEBS Journal, DOI: 10.1111/febs.l5477).
  • DNA-binding dyes are known in the art. DNA-binding dyes include, but are not limited to, DAPI (4’,6-diamidino-2-phenylindole), 7-AAD (7- aminoactinomycin D), Hoechst 33342, Hoechst 33258, Hoechst 34580, intercalators comprising a lanthanide chelate, and cyanine dyes such as SYBR GreenTM and PicoGreenTM.
  • Hoechst 33342 a double-stranded DNA-binding dye
  • JF549 can be conjugated to JF549 to label DNA with JF549.
  • the DNA-Hoechst 33342 -JF549 conjugate JF549-labelled second molecule
  • JF646-labeled molecule can be used to selectively photoactivate a JF646-labeled molecule that binds to the DNA.
  • a conjugate of an effector fluorophore or a rhodamine dye to a lipid such as cholesterol can be used.
  • JF549 can be conjugated to cholesterol (or other membrane lipid); and the JF-549-lipid conjugate can be used to selectively photoactivate a JF646-labeled molecule that is associated with a membrane.
  • a subject method comprises detecting fluorescence from the reactivated rhodamine dye in the contacted sample. Detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule were within sufficient proximity of one another (e.g., from about 0.1 nm to about 25 nm from one another, as described above) in the contacted sample when the effector fluorophore was excited. Fluorescence from the reactivated rhodamine dye can be detected using any of a variety of known techniques and instrumentation. For example, fluorescence can be detected using a fluorescence microscope, a fluorometer, a fluorescence scanner, or a flow cytometer.
  • a subject method further comprises normalization of fluorescence, e.g., by exposing the sample (comprising the first molecule comprising the inactivated rhodamine dye and the second molecule comprising the effector fluorophore) to light of a wavelength that directly reactivates the inactivated rhodamine dye independent of proximity to the effector dye.
  • a subject method further comprises normalization of fluorescence, e.g., by exposing the sample (comprising the first molecule comprising the inactivated rhodamine dye and the second molecule comprising the effector fluorophore) to light of a wavelength that directly reactivates the inactivated rhodamine dye independent of proximity to the effector dye.
  • proximity-independent reactivation of the inactivated rhodamine dye can serve as a quantitative internal standard to which proximity-dependent reactivation of the inactivated rhodamine dye may be normalized.
  • the JF646 can be inactivated by exposure to light of 633 nm, thereby generating a sample (an “inactivated sample”) comprising a first molecule comprising inactivated JF646.
  • Exposure of the inactivated sample to light of a wavelength of less than 500 nm e.g., from about 300 nm to about 400 nm, from about 400 nm to about 495 nm, from about 400 nm to about 450 nm, or from about 405 nm to about 488 nm
  • a wavelength of less than 500 nm e.g., from about 300 nm to about 400 nm, from about 400 nm to about 495 nm, from about 400 nm to about 450 nm, or from about 405 nm to about 488 nm
  • exposure of the inactivated sample to light of a wavelength of from about 405 nm to about 488 nm reactivates the JF646 in a proximity-independent manner. In some cases, exposure of the inactivated sample to light of a wavelength of about 488 nm reactivates the JF646 in a proximity-independent manner.
  • a suitable effector fluorophore is one that reactivates the inactivated rhodamine dye (that is linked to the first molecule) in a proximity-dependent manner; i.e., the effector fluorophore is linked to the second molecule (directly or via a linker) and reactivates that inactivated rhodamine dye (that is linked to the first molecule) only when the first molecule and the second molecule are in sufficiently close proximity to one another (as described above).
  • a suitable effector fluorophore is JF549, which emits at a range of wavelengths including 561 nm and 590 nm.
  • a second molecule comprising JF549 can, when in sufficient proximity to a first molecule comprising JF646 that has been inactivated by exposure to light of 633 nm, reactivate the inactivated JF646, even though JF646 is not directly reactivated by exposure to light of either 561 nm or 590 nm.
  • an inactivated sample (a sample comprising: i) a first molecule comprising a rhodamine dye that has been inactivated by exposure to light of an inactivating wavelength; and ii) a second molecule comprising an effector fluorophore) is exposed to: a) light of a wavelength that excites the effector fluorophore, such that the effector fluorophore reactivates the inactivated rhodamine dye in a proximity-dependent manner; and b) light of a wavelength that directly reactivates the inactivated rhodamine dye in a proximity-independent manner.
  • the exposure to the (a) light of a wavelength that excites the effector fluorophore, such that the effector fluorophore reactivates the inactivated rhodamine dye in a proximity-dependent manner; and (b) light of a wavelength that directly reactivates the inactivated rhodamine dye in a proximity-independent manner can be carried out simultaneously.
  • the exposure to the (a) light of a wavelength that excites the effector fluorophore, such that the effector fluorophore reactivates the inactivated rhodamine dye in a proximity-dependent manner; and (b) light of a wavelength that directly reactivates the inactivated rhodamine dye in a proximity-independent manner can be carried out at different times, e.g., in an alternating manner.
  • the ratio of the fluorescence from proximity-dependent reactivation of the inactivated rhodamine dye to the fluorescence from proximity-independent reactivation of the inactivated rhodamine dye can indicate what fraction of molecules labeled with the rhodamine dye are in proximity to molecules labeled with the effector fluorophore.
  • the difference in proximity-dependent and proximity-independent reactivation can be used as a measure of proximity.
  • a proximity-dependent photoactivation method of the present disclosure is useful for a variety of research and diagnostic applications.
  • a method of the present disclosure can be used for live cell single-molecule imaging, super-resolution imaging, and related imaging-based methods.
  • a proximity-dependent photoactivation method of the present disclosure can be used to detect binding of two molecules (a first molecule and a second molecule; such as two polypeptides, a polypeptide and a polynucleotide, etc., as described above) in a living cell in vitro.
  • the effect of various compounds and/or conditions on the binding of the two molecules to one another can be determined using a proximity-dependent photoactivation method of the present disclosure.
  • binding of a first molecule to a second molecule can be carried out in the presence of a test compound (e.g., a small molecule (e.g., a drug), a cytokine, a chemokine, a ligand for a cell-surface receptor, and the like); and the effect of the test compound on binding of the first molecule to the second molecule can be determined by comparing the binding in the presence of the test compound to the binding in a control sample which does not include the test compound (i.e., binding in the absence of the test compound).
  • a test compound e.g., a small molecule (e.g., a drug), a cytokine, a chemokine, a ligand for a cell-surface receptor, and the like
  • the effect of the test compound on binding of the first molecule to the second molecule can be determined by comparing the binding in the presence of the test compound to the binding in a control sample which does not include the test compound (i.e.,
  • the effect of a condition on the binding of a first molecule to a second molecule can be tested by determining the effect of the condition on the binding, and comparing the binding in the presence of the condition to the binding in the absence of the condition.
  • Such conditions can include, e.g., pH, temperature, osmolarity, contacting the cell comprising the first and second molecules to a second cell (cell-cell contact), and the like.
  • association of a protein with a cell membrane can be identified using a proximity-dependent photoactivation method of the present disclosure.
  • a conjugate of an effector fluorophore or a rhodamine dye to a lipid such as cholesterol can be used.
  • a cell membrane can be labeled by incorporation into the cell membrane of an effector fluorophore-labeled membrane lipid.
  • the effector fluorophore-lipid conjugate integrates into the cell membrane, and can reactivate an inactivated rhodamine dye that is conjugated to a first molecule when the first molecule binds to the membrane.
  • JF549 can be conjugated to cholesterol (or other membrane lipid); and the JF549-lipid conjugate can be incorporated into a cell membrane, where it can selectively photoactivate a JF646-labeled molecule (e.g., a JF646-labeled protein) that is associated with a membrane.
  • a JF646-labeled molecule e.g., a JF646-labeled protein
  • labeling of a small molecule drug with an effector fluorophore can be used to selectively photoactivate a rhodamine-dye-labeled protein, where the rhodamine dye has been inactivated, to determine binding of the drug to the protein.
  • a proximity-dependent photoactivation method of the present disclosure can be used to detect post-translational modification of a protein with a modifying group by labeling the modifying group with an effector fluorophore and the protein with a rhodamine dye.
  • an effector fluorophore -labeled sugar can be used to detect post- translational glycosylation of a rhodamine dye-labeled protein.
  • an effector fluorophore-labeled HALO tag-ubiquitin fusion protein could be used to detect ubiquitinylation of rhodamine dye-labeled protein.
  • a proximity-dependent photoactivation method of the present disclosure can be used in combination with a super-resolution imaging method to reveal both three-dimensional structure and molecular interactions within cells.
  • a proximity-dependent photoactivation method of the present disclosure can be used to detect aggregation of labeled proteins, including small oligomeric aggregates that are not clearly visible by other means.
  • Small oligomers may contribute to the pathological effects of aggregation-prone proteins (such as amyloid beta in Alzheimer's disease) and presage the formation of larger aggregates.
  • a system of the present disclosure can comprise: a) a first molecule that comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore; b) a second molecule that comprises the effector fluorophore; and c) a light source that is configured to produce the first wavelength of light that inactivates a rhodamine dye. In some cases, the light source is also configured to produce a wavelength of light that excites the effector fluorophore.
  • a method of detecting proximity of a first molecule to a second molecule comprising: a) contacting the first molecule with the second molecule to form a contacted sample, wherein: i) the first molecule comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore in a proximity-dependent manner; and ii) the second molecule comprises the effector fluorophore, and wherein said contacting is carried out under conditions that inactivate the rhodamine dye; b) exciting the effector fluorophore with a second wavelength of light, wherein the excited effector fluorophore reactivates the inactivated rhodamine dye; and c) detecting fluorescence from the reactivated rhodamine dye in the contacted sample, wherein fluorescence from the reactivated rhodamine dye indicates that the first molecule
  • Aspect 2 The method of aspect 1, wherein the rhodamine dye is inactivated at 633 nm.
  • Aspect 3 The method of aspect 1 or aspect 2, wherein the rhodamine dye is a silicon rhodamine dye, optionally wherein the rhodamine dye is Janelia Fluor 646, Janelia Fluor X 646 or Janelia Fluor X 650.
  • Aspect 4 The method of aspect 1 , wherein the effector fluorophore is a second rhodamine dye or a fluorescent polypeptide.
  • Aspect 5 The method of aspect 4, wherein the effector fluorophore is JF549, JFX549, JF526, Alexa Fluor 532, Alexa Fluor 549, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, tetramethylrhodamine, ATTO 532, ATTO Rho6G, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol l, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO590, ATTO Rhol3, or ATTO594.
  • the effector fluorophore is JF549, JFX549, JF526, Alexa Fluor 532, Alexa Fluor 549, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, tetramethylrhodamine, ATTO 532, ATTO Rho6G, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol
  • Aspect 6 The method of any one of aspects 1-5, wherein the first molecule is a biomolecule.
  • Aspect 7 The method of any one of aspects 1-6, wherein the second molecule is a biomolecule.
  • Aspect 8 The method of aspect 6 or aspect 7, wherein:
  • the first biomolecule is selected from the group consisting of a nucleic acid, a lipid, a carbohydrate, a small molecule, and a polypeptide;
  • the second biomolecule is selected from the group consisting of a nucleic acid, a lipid, a carbohydrate, a small molecule, and a polypeptide.
  • Aspect 9 The method of any one of aspects 6-8, wherein the first biomolecule is a polypeptide and wherein the second biomolecule is a polypeptide.
  • Aspect 10 The method of any one of aspects 6-8, wherein:
  • the first biomolecule is a nucleic acid and wherein the second biomolecule is a polypeptide
  • the first biomolecule is a polypeptide and the second biomolecule is a nucleic acid;
  • the first biomolecule is a nucleic acid and the second biomolecule is a nucleic acid.
  • Aspect 11 The method of any one of aspects 6-8, wherein:
  • the first biomolecule is a lipid and wherein the second biomolecule is a polypeptide
  • the first biomolecule is a polypeptide and the second biomolecule is a lipid.
  • Aspect 12 The method of any one of aspects 6-8, wherein:
  • the first biomolecule is a carbohydrate and wherein the second biomolecule is a polypeptide
  • the first biomolecule is a polypeptide and the second biomolecule is a carbohydrate.
  • Aspect 13 The method of any one of aspects 1-10, wherein the first molecule is a fusion polypeptide that comprises a heterologous fusion polypeptide selected from a SNAP, a CLIP, a HALO polypeptide, a dihydrofolate reductase (DHFR) polypeptide, a tetracysteine tag, a sortase polypeptide, or a transglutaminase polypeptide, and wherein the rhodamine dye is linked to the first molecule via the SNAP, CLIP, HALO polypeptide, DHFR polypeptide, tetracysteine tag, sortase polypeptide, or transglutaminase polypeptide.
  • a heterologous fusion polypeptide selected from a SNAP, a CLIP, a HALO polypeptide, a dihydrofolate reductase (DHFR) polypeptide, a tetracysteine tag, a sort
  • Aspect 14 The method of any one of aspects 1-10, wherein the second molecule is a fusion polypeptide that comprises a heterologous fusion polypeptide selected from a SNAP, a CLIP, or a HALO polypeptide, and wherein the effector fluorophore is linked to the first molecule via the SNAP, CLIP, or HALO polypeptide.
  • Aspect 15 The method of any one of aspects 1-14, wherein the first molecule and the second molecule are in vitro outside of a cell.
  • Aspect 16 The method of any one of aspects 1-14, wherein the first molecule and the second molecule are present in a cell in vitro.
  • Aspect 17 The method of any one of aspects 1-14, wherein the first molecule and the second molecule are present in a cell in vivo.
  • Aspect 18 The method of aspect 16 or aspect 17, wherein the cell is a eukaryotic cell.
  • Aspect 19 The method of aspect 18, wherein the cell is present in a tissue or an organ.
  • Aspect 20 The method of any one of aspects 1-19, wherein said detecting is carried out using a microscope.
  • Aspect 21 The method of any one of aspects 1-20, further comprising reactivating the inactivated rhodamine dye by exposing the contacted sample to light of a wavelength of less than 500 nm, thereby activating the rhodamine dye in a proximity-independent manner.
  • Aspect 22 The method of aspect 21, comprising reactivating the inactivated rhodamine dye by exposing the contacted sample to light of a wavelength of from about 405 nm to about 488 nm.
  • a system comprising:
  • a light source that is configured to produce the first wavelength of light that inactivates a rhodamine dye.
  • Aspect 24 The system of aspect 23, wherein the light source is configured to produce a wavelength of light that excites the effector fluorophore.
  • Aspect 25 The system of aspect 23 or aspect 24, wherein:
  • the rhodamine dye is a silicon rhodamine dye, optionally wherein the rhodamine dye is Janelia Fluor 646, Janelia Fluor X 646 or Janelia Fluor X 650; and
  • the effector fluorophore is a second rhodamine dye or a fluorescent polypeptide, optionally wherein the effector fluorophore is JF549, JFX549, JF526, Alexa Fluor 532, Alexa Fluor 549, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, tetramethylrhodamine, ATTO 532, ATTO Rho6G, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol l, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO590, ATTO Rhol3, or ATTO594.
  • the effector fluorophore is JF549, JFX549, JF526, Alexa Fluor 532, Alexa Fluor 549, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, tetramethylrhodamine, ATTO 532, ATTO Rho6G,
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second( s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
  • U2OS cells with a Cas9 HDR-generated HaloTag fusion to the endogenous NPM1 gene were cultured in Dulbecco’s modified Eagle medium (DMEM). Cells were stained for approximately 20 minutes at 37°C with either 50 pM JFX650 HaloTag ligand alone in DMEM or a mixture of 50 pM JFX650 HaloTag ligand and 10 nM JF549 HaloTag ligand in DMEM. After staining, cells were washed twice with lx phosphate buffered saline, and destained for 15 minutes in DMEM at 37°C before imaging.
  • DMEM Dulbecco’s modified Eagle medium
  • HILO highly inclined and laminated optical sheet
  • the imaging protocol consisted of 500-frame intervals of illumination with a 633 nm laser alternating with 60-frame intervals of illumination with a 561 nm laser.
  • Particletracking software was used to count single molecule localizations, and the number of localizations at each frame index was averaged over multiple cells.
  • U2OS cells were cultured in Dulbecco’s modified Eagle medium (DMEM). Half of the cells from a 10-cm culture dish were transiently transfected using the Lonza Nucleofector kit with 500 ng of an expression plasmid encoding either 1) a HaloTag fusion of a monomeric single-chain derivative of the bacterial Tet repressor (Halo-scTetR; Fig. 3 A; Kamionka et al., 2006, Nucleic Acids Research, DOI: 10.1093/nar/gkl316) or 2) a HaloTag fusion of dimeric Tet repressor (Halo-TetR; FIG. 3B).
  • DMEM Dulbecco’s modified Eagle medium
  • Halo-TetR protein dimers have two HaloTag moieties each, while Halo-scTetR protein monomers have only a single HaloTag moiety each.
  • DMEM phosphate-buffered saline
  • HILO highly inclined and laminated optical sheet
  • the imaging protocol consisted of 500-frame intervals of illumination with a 633 nm laser alternating with 5-frame intervals of illumination with either a 488 nm laser or 561 nm laser.
  • Particle-tracking software was used to count single molecule localizations, and the number of localizations at each frame index was averaged over multiple cells.
  • FIG. 3 shows the mean number of single-molecule JF646 localizations in each frame, averaged over multiple experimental replicates. Illumination at 633 nm was interspersed with brief 5-frame pulses of 488 nm light (black arrows; FIG. 3) or 561 nm light (grey arrows; FIG. 3). Illumination with 561 nm light (grey arrows and plot segments) reactivated JF646 for Halo-TetR dimers but not for Halo-scTetR monomers.
  • FIG. 6A-6C depict multiple cycles of alternating illumination.
  • FIG. 6A depicts a scheme for quantification of the rate of proximity-dependent reactivation (e.g., by JF549 + 561 nm light), using direct reactivation of JF646 fluorophores with a shorter wavelength (e.g., 405 nm or 488 nm) as an internal standard.
  • Open bar 633 nm. The sample was imaged using 633 nm light and alternately stimulated with 405 or 488 nm light (to directly reactivate JF646) and 561 nm light (to induce proximity-dependent reactivation).
  • FIG. 6A depicts a scheme for quantification of the rate of proximity-dependent reactivation (e.g., by JF549 + 561 nm light), using direct reactivation of JF646 fluorophores with a shorter wavelength (e.g., 405 nm or 488 nm) as an internal standard.
  • FIG. 6B depicts a trace showing changes in the intensity of a sample in response to successive 561 nm (grey) and 488 nm (black) pulses.
  • FIG. 6C A rate constant can be determined by varying the duration of the green pulse (depicted in FIG. 6B as grey), while keeping the duration of the blue pulse (depicted in FIG. 6B as black) constant at 7 milliseconds (ms).
  • the plot of reactivation ratio vs. green pulse duration is well fit by a single-exponential curve, consistent with first-order kinetics.
  • U2OS cells transfected with a TetR-Halo expression plasmid (as in Fig. 3) were fixed for 20 min at room temperature with 4% paraformaldehyde in lx phosphate buffered saline (lx PBS). Cells were rinsed twice with lx PBS, permeabilized for 45 min with 0.1% Triton X-100 in lx PBS, washed twice more with lx PBS, and stored overnight at 4°C. Fixed permeabilized cells were imaged at a frame rate of 7.5 ms/frame using HILO illumination, alternating between 500 frames of 633 nm illumination and 5 frames of 561 nm illumination. Single-molecule localizations were counted using particle tracking software, and the number of localizations at each frame index was averaged over multiple cells.
  • U2OS cells were electroporated with an expression plasmid encoding a fusion between the HaloTag and SNAPf tag proteins, connected by a flexible linker peptide with sequence GSGTGS.
  • Cells were stained with 5 nM JFX650 SNAP ligand and 50 nM JF549 Halo ligand for 15 min at 37°C, washed twice with lx PBS, and destained for 15 min in DMEM.
  • Cells were imaged with highly inclined and laminated optical sheet (HILO) illumination on an inverted Nikon TIRF microscope at a frame rate of 7.5 ms/frame.
  • HILO highly inclined and laminated optical sheet
  • the imaging protocol consisted of 500- frame intervals of illumination with a 633 nm laser alternating with 1 -frame intervals of illumination with either a 488 nm laser (black in FIG. 5) or 561 nm laser (grey in FIG. 5).
  • Particle-tracking software was used to count single molecule localizations, and the number of localizations at each frame index was averaged over multiple cells.
  • a Halo-SNAP fusion protein was labeled with a mixture of JFX650 SNAP ligand and JF549 HaloTag ligand.
  • SNAP-bound JFX650 was efficiently reactivated by 561 nm light in the presence of Halo-bound JF549.
  • a combination of HaloTag and SNAP tag labeling can be used in the proximity-dependent photoactivation method.
  • FIG. 8 depicts application of proximity-dependent reactivation to detect inducible dimerization of JF549-labeled FRB-Halo and JFX650-labeled FKBP-SNAPf.
  • U2OS cells were co-transfected with expression plasmids encoding the two proteins, labeled with JF549 Halo ligand and JFX650 SNAP ligand, and imaged using the alternating excitation protocol, either before or after addition of 333 nM rapamycin, which induces FRB-FKBP dimerization.
  • An increase in the AGreen/ABlue (AGrey/ABlack) reactivation ratio was seen following rapamycin addition (shown are three experimental replicates).
  • FIG. 9 depicts application of proximity-dependent reactivation to detect inducible dimerization of androgen receptor (AR) double-labeled with JF549 and JFX650.
  • AR androgen receptor
  • U2OS cells were co-transfected with expression plasmids encoding the Halo- AR and SNAPf-AR, labeled with JF549 Halo ligand and JFX650 SNAP ligand, and imaged using the alternating excitation protocol.
  • Dihydrotestosterone (DHT) was added to a final concentration of 10 nM immediately after imaging cell #11. An increase in the AGreen/ABlue (AGrey/ABlack) reactivation ratio was seen over time as successive cells were imaged after DHT addition.
  • FIG. 10 depicts displacement histograms for 561 nm-reactivated, 405 nm-reactivated, and spontaneously reactivated molecules from the previous experiment (depicted in FIG. 9). Single molecules were tracked, and the resulting trajectories were analyzed using the Spot-On analysis package (Hansen et al. (2016) Elife 7:c33125) to infer the fraction of freely diffusing and bound molecules. The calculated bound fraction was substantially higher among 561 nm- reactivated molecules, consistent with a higher propensity of self-associated AR molecules to bind DNA than the overall mixture of monomeric and multimeric AR molecules.
  • FIG. 11A-11B show that proximity-dependent photoactivation can be used to detect differences in the bound fraction of distinct molecular subpopulations within the same cell.
  • U2OS cells were transfected with a dual expression plasmid encoding a mixture of either A) Halo-SNAPf-tagged histone H2B (mostly bound) and SNAPf-3xNLS (mostly unbound; “3xNLS” denotes three repeats of the minimal SV40 nuclear localization sequence) or B) SNAPf-tagged histone H2B and SNAPf-Halo-3xNLS.
  • Cells were labeled with JF549 Halo ligand and JFX650 SNAP ligand.
  • Each cell was illuminated with 633 nm light to place JFX650 in a dark state and then imaged with 561 nm reactivation pulses followed by 405 nm reactivation pulses. Pairs of points show the bound fraction of 561 nm-reactivated and 405 nm-reactivated molecules for each cell. Molecules reactivated by 561 nm light have a higher bound fraction in (A) and a lower bound fraction in (B), consistent with selective reactivation of the doublelabeled component by 561 nm light.
  • FIG. 12 presents data indicating that proximity-dependent reactivation occurs over long distances. Insertion of a 67-amino acid rigid helical spacer between Halo and SNAPf led to little reduction in the reactivation of JFX650 by JF549. Because this spacer ostensibly contributes an additional 100-angstrom separation between Halo and SNAPf, this result suggests that the distance dependence of proximity-dependent photoactivation may be looser than that of singlemolecule FRET.
  • PAPA proximity-assisted photoactivation
  • FRET Forster resonance energy transfer
  • PAPA can be used in live cells both to detect protein-protein interactions and to enrich for a sub-population of labeled protein complexes in which two different dyes are in proximity.
  • PAPA detected the expected correlation between androgen receptor self-association and chromatin binding within individual cells.
  • JFX650 Janelia Fluor X 650
  • JF549 Janelia Fluor 549
  • PAPA proximity-assisted photoactivation
  • PAPA of JF dyes occurs under physiological conditions in live cells and requires neither an oxygen scavenging system nor high concentrations of exogenous thiols. While PAPA requires proximity between the two fluorophores, its effective distance range extends beyond that of FRET, making it potentially a more versatile approach.
  • PAPA provides a way to detect protein complexes in live cells. It is shown herein that PAPA can be used to detect the formation of protein dimers and that it can selectively highlight sub-populations of molecules within defined mixtures. As a further proof of concept, singlemolecule tracking was combined with PAPA to analyze the increase in chromatin binding induced by self-association of androgen receptor. By enabling the previously elusive detection of protein-protein interactions, PAPA provides expanded information for live cell single-molecule imaging.
  • U2OS cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose (ThermoFisher # 10566016), 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin at 37°C and 5% CO2. Phenol red-containing medium was used for propagation of cells, while phenol red-free medium (ThermoFisher # 21063029) was used to minimize fluorescence background in imaging experiments.
  • Ig linkers were derived from a previously reported plasmid containing repeats of the titin 191 Ig domain, which were codon-shuffled to prevent recombination (Scholl et al. (2016) Biomacromolecules 17:2502).
  • U2OS cells from a confluent 10-cm plate were trypsinized, resuspended in DMEM, and divided between two 15-ml conical tubes. Cells were centrifuged for 2 min at 200 g, and the medium was aspirated and replaced with 100 pl of Lonza Kit V transfection reagent (82 pl of Kit V solution and 18 pl of Supplement I) containing 1 pg of the donor plasmid and 1 pg of Super PiggyBac transposase plasmid. The cell suspension was transferred to an electroporation cuvette and electroporated using program X- 001 on the Amaxa Nucleofector II (Lonza).
  • Cells in the cuvette were mixed with 300 pl of DMEM, and 100 pl of the suspension was plated in a 10-cm plate. After allowing cells to grow for 1-2 days, selection was initiated by adding puromycin to a final concentration of 1 pg/ml.
  • Cells expressing pTG820 (Halo-3x Ig-SNAPf) and pTG828 (Halo-5x Ig-SNAPf) were sorted on a different day using the intensity of the previously sorted pTG747/U2OS pool to define a gate in the JFX650- Halo channel.
  • Lysates were prepared by addition of 1 ml (for 10-cm plates) or 200 pl (for 6-well plates) of sodium dodecyl sulfate (SDS) lysis buffer without loading dye (Cattoglio et al. (2019), doi:10.7554/eLife.40164). Each lysate was passed through a 26-gauge needle 10 times to reduce its viscosity.
  • SDS sodium dodecyl sulfate
  • FIG. 20A-20F FIG. 26A-26D, FIG. 25A-25F, FIG. 14B-14D, FIG. 19B, FIG. 22A-22H, FIG. 23A-23I, and FIG. 24B-24C;
  • Coverslips were mounted in a stainless steel AttofluorTM Cell Chamber (ThermoFisher #A7816) and covered with 1 ml of phenol red-free DMEM with 10% FBS and penicillin/streptomycin. Cells were imaged using near-TIRF illumination on the microscope described in detail in [Hansen AS et al., Elife, 2018, PMID 29300163]. Laser power densities used for imaging were approximately 52 W/cm 2 for 405 nm, 100 W/cm 2 for 561 nm, and 2.3 kW/cm 2 for 633 nm. Fluorescence emission was filtered through a Semrock 676/37 bandpass filter.
  • FIG. 13A-13B, FIG. 19B, and FIG. 24B 10 cycles of 250 R [2 ms], 1 V [7 ms], 500 R [2 ms], 1 G [7 ms], 250 R [2 ms]
  • FIG. 13C and FIG. 17A-17B 5 cycles of 100 R [7 ms], 1 V [7 ms] + R [7 ms], 200 R [7 ms], 1 G [7 ms] + R [7 ms], 100 R [7 ms]
  • FIG. 18A-18C Same as in FIG. 13A-13B, but only 4 cycles.
  • FIG. 20A-20F, FIG. 22A-22D, and FIG. 23A-23F Cells were first illuminated 10 s with 633 nm light to either photobleach or shelve most JFX650 fluorophores and then imaged with 10 cycles of 250 R [2 ms], 1 V [0.5 ms] + R [2 ms], 500 R [2 ms], 1 G [2 ms] + R [2 ms], 250 R [2 ms].
  • FIG. 25A-25F, FIG. 22E-22H, and FIG. 23G-23I Same as FIG. 20A-20F, but with green pulses shortened to 0.5 ms.
  • FIG. 26B and FIG. 24B Same as FIG. 13A-13B, but only 5 cycles.
  • FIG. 26C Same as FIG.20A-20F, but with green pulses extended to 7 ms.
  • FIG. 26D Same as FIG.20A-20F.
  • FIG. 14A 10 cycles of 100 R [7 ms], 1 V [7 ms] + R [7 ms], 100 R [7 ms]. Only the first two cycles are shown in the figure.
  • FIG. 24C Same as FIG. 20A-20F with green pulse duration set to 7 ms before DHT addition and 2 ms after DHT addition.
  • DHT addition increases the rate of PAPA, i.e., the number of fluorophores reactivated by a green pulse of a given duration (FIG. 26B).
  • the green pulse duration was thus shortened after DHT addition to keep the number of localizations per frame roughly equivalent and to prevent PAPA trajectories from becoming too dense for accurate tracking.
  • the total pixel intensity was summed for all 20 frames in phases (1), (3), and (5), and the fractional reactivation was calculated by subtracting the increase in signal between (3) and (5) by the initial drop in signal between (1) and (3).
  • the number of violet frames in phase 4, N was varied as indicated by the values on the horizontal axis of FIG. 14B. The data were fitted to a singleexponential model.
  • Fluorescence lifetime was measured on a Zeiss LSM 980 microscope equipped with a Becker-Hickl SPC-150NX TCSPC module.
  • Cells were labeled with a mixture of 50 nM JFX650- HTL and 50 nM JF549-STL for 1 h at 37°C, or with 50 nM JF549-STL alone as a no-FRET control.
  • Particles were localized and tracked using the quot package (https(://)github(dot)com/ alecheckert/quot).
  • Custom MATLAB code (GitLab URL) was used to extract all trajectory segments occurring between 2 and 31 frames after pulses of 405 nm light (DR trajectories) and 561 nm light (PAPA trajectories). PAPA and DR trajectories were then separately analyzed using a Bayesian “fixed-state sampler” algorithm (https://github.com/alecheckert/spagl) (Heckert, et al.
  • FIG. 25F, and FIG. 231 by fitting to a 3-state model with diffusion coefficients 0.01, 2.1, and 13.2 p nr/s (FIG. 25C) or 0.01, 1.3, and 15.8 pm 2 /s (FIG. 25F and FIG. 231).
  • Fraction bound for androgen receptor (FIG. 35C) was calculated by fitting to a 2-state model with diffusion coefficients 0.01 and 4.4 m 2 /s. Diffusion coefficients used in the reduced models correspond to the local maxima of the ensemble distributions.
  • Displacement histograms in FIG. 22B, 22D, 22F, and 22H were tabulated using custom code in MATLAB.
  • PAPA Proximity-assisted photoactivation
  • PAPA was observed while imaging an oligomeric protein labeled with two different JF dyes.
  • U2OS cells expressing Halo-tagged NPM1 (a pentameric nucleolar protein) (Heckert et al. (2021) BioRxiv, doi.org/10.1101/2021.05.03.442482) were labeled with a low concentration of Janelia Fluor X 650 Halo-tag ligand (JFX650-HTL)(20) to track single molecules, together with a higher concentration of Janelia Fluor 549 HaloTag ligand (JF549-HTL) to visualize nucleoli.
  • JFX650-HTL Janelia Fluor X 650 Halo-tag ligand
  • JFX650 When JFX650 was excited with red light (633 nm) and, alternately, JF549 was excited with green light (561 nm), it was noticed that some JFX650 molecules that had gone dark during red illumination suddenly reappeared after green illumination (green vertical lines in FIG. 13A (top panel) and FIG. 13B and green box in FIG. 13B). Reactivation of JFX650 by violet light was observed, both with and without JF549-HTL (violet vertical lines in FIG. 13A (top panel; middle panel) and violet box in FIG. 13B). However, reactivation of JFX650 by green light required co- labeling with JF549 (compare FIG.
  • FIG. 13A-13C Proximity-assisted photoactivation (PAPA) of JFX650 by JF549.
  • A Average number of localizations in the JFX650 channel as a function of frame number for heterozygous NPMl-Halo U2OS cells labeled with different combinations of JFX650-HTL and/or JF549-HTL. JFX650 molecules were excited with 633 nm light, interspersed with 7 ms pulses of 405 nm and 561 nm light (violet and green vertical lines).
  • Right column Schematic of NPM/NPMl-Halo pentamers labeled with JF549 (orange) and/or JFX650 (red).
  • FIG. 14A-14D Properties of JFX650 reactivation.
  • A) Comparison of darkening and reactivation of JFX650 bound to Halo (cyan) and SNAPf (black). Relative fluorescence intensity is plotted on the y-axis, averaged over multiple cells (n 10 for Halo, 14 for SNAPf), and frame number is plotted on the x-axis. The frame rate was 7 ms/frame. Fluorescence intensity declined more rapidly for SNAPf-JFX650 than for Halo-JFX650.
  • Violet pulses of 7 ms at frames 101 and 302 induced direct reactivation of JFX650, which was greater for JFX650-SNAPf than for JFX650-Halo.
  • B Reactivation of SNAPf-JFX650 as a function of 405 nm pulse duration.
  • SNAPf-JFX650 intensity was measured three times using 20 frames of 1 ms stroboscopic 633 nm illumination: 1) before darkening, 2) after darkening with 400 frames of non-stroboscopic (7 ms/frame) illumination, and 3) after reactivation by exposure to 405 nm pulses of varying duration.
  • Percent reactivation was calculated by dividing the increase in intensity after reactivation by the decrease in intensity after darkening. Solid black curve shows a fit to a single-exponential model (black curve).
  • FIG. 15A-15E PAPA between other sender-receiver pairs.
  • U2OS cells expressing Halo-SNAPf-3xNLS were labeled with different SNAP tag ligand (STL) and HaloTag ligand (HTL) fluorophore combinations and imaged with red (633 nm) light alternating with 7-ms pulses of green (561 nm) and violet (405 nm) light. Fluorescence intensity averaged over multiple cells is plotted on the vertical axis and frame number is plotted on the horizontal axis.
  • TMR Tetramethylrhodamine
  • JFX650-STL JFX650-STL.
  • FIG. 16A-16B SDS-PAGE analysis of linker constructs.
  • A-B SDS-PAGE analysis of linker constructs expressed in U2OS cells and labeled with JF549-HTE (A) or JF549-STE (B). The amount of cell lysate loaded in each lane corresponds to 100,000 cells.
  • Eookup table is set between 0 and 3000 counts for (A) and the top image in (B).
  • the bottom image in (B) is the same gel with lookup table set between 0 and 800 counts to highlight faint bands.
  • MW molecular weight markers in kilodaltons. *, nonspecific bands. 0, unbound dye.
  • a ladder of smaller fragments is present below the full-length protein for the larger linkers; these are predominantly labeled by Halo ligand but not SNAP ligand (see Note 1).
  • FIG 17A-17B Comparison of distance-dependence of PAPA and FRET.
  • B Eeft panel: Reactivation rate of PAPA (slope of fits in (a) divided by the slope of the short linker construct).
  • Right panel FRET efficiency measured using fluorescence lifetime imaging (FEIM). Detection of inducible protein-protein interactions using PAPA
  • FIG. 18A-18C Detection of inducible dimerization using PAPA.
  • Halo-FRB was labeled with the sender fluorophore (JF549) and SNAPf-FKBP with the receiver fluorophore (JFX650). After shelving JFX650 with red (633 nm) light, DR and PAPA were alternately induced with pulses of violet (405 nm) and green (561 nm) light, respectively. Midway through the experiment, cells were treated with rapamycin to induce FRB-FKBP dimerization or with dimethylsulfoxide (DMSO) solvent as a negative control. (B).
  • DMSO dimethylsulfoxide
  • C Average PAPA/DR ratio before (-) and after (+) addition of rapamycin (Rapa) or DMSO. Total number of cells: 75 before and 74 after rapamycin, 30 before and 30 after DMSO. Error bars, ⁇ 2*SEM. Statistical significance was calculated using a 2-tailed t-test.
  • FIG. 19A-19B SDS-PAGE and PAPA traces of FRB-FKBP.
  • PAPA optically enriches a subset of molecules in defined 2-component mixtures
  • SNAPf-H2B SNAPf-tagged histone H2B
  • Halo-SNAPf- 3xNLS nuclearly localized Halo-SNAPf fusion
  • FIG. 20A Cells were incubated with a mixture of JFX650-STL and JF549-HTL to label SNAPf-H2B with JFX650 only and Halo-SNAPf-3xNLS with both JFX650 and JF549 (FIG. 20A; FIG. 21A).
  • JFX650 fluorophores were thoroughly photobleached/shelved using a 10-s pulse of intense red light, after which JFX650 was imaged with red light interspersed with pulses of green and violet light.
  • PAPA trajectories those occurring after a green pulse
  • DR trajectories those occurring after a violet pulse
  • saSPT Bayesian state array SPT
  • PAPA trajectories were enriched for freely diffusing molecules compared to DR trajectories, as expected if PAPA selectively reactivates JF549/JFX650 doublelabeled Halo-SNAPf-3xNLS molecules (FIG. 20B).
  • FIG. 22C-22D As a further control, cells expressing either Halo-SNAPf-3xNLS or Halo- SNAPf-H2B alone were analyzed; as expected, PAPA and DR trajectories displayed virtually identical diffusion spectra for single components (FIG. 23A-23F).
  • Cytosolic Halo- SNAPf was co-expressed with a SNAPf-tagged synthetic protein that forms large, slowly diffusing 60-mers (Shia et al. (2016) Nature 535:136) (FIG. 25A), and imaging and analysis were performed as described above. Diffusion spectra showed the expected mixture of slow- diffusing (SNAPf-60-mer) and fast-diffusing (Halo-SNAPf) subpopulations (FIG. 25B).
  • PAPA trajectories (green curve) were strongly enriched in the fast-diffusing subpopulation, consistent with selective reactivation by green light of the double-labeled Halo-SNAPf protein (FIG. 25B).
  • the same trend was observed in individual cells (FIG. 25C) and was apparent from comparison of displacement histograms and raw trajectories (FIG. 22E-22F).
  • the enrichment of the fast-diffusing population was not absolute, as a slow-diffusing peak was still observed among PAPA trajectories (FIG. 25B, solid black curve).
  • FIG. 20A-20F “Unmixing” of defined 2-component mixtures using PAPA.
  • Left column (a,d) Schematic of different defined mixtures of two labeled proteins, in which one protein is labeled with JFX650 only and the other is labeled with both JFX650 and JF549.
  • Center column (b,e) Inferred diffusion spectra of PAPA (green-reactivated) and DR (violet-reactivated) trajectories pooled from 20 cells.
  • Right column (c,f): Fraction bound of PAPA and DR trajectories from individual cells, obtained from fits to a 2-state model. Paired, two-tailed t-tests of the comparisons in (c) and (f) showed both differences to be statistically significant with p 6 x 10 9 and 7 x 10 8 , respectively.
  • FIG. 21A-21B SDS-PAGE gels of defined 2-component mixtures and 1-component controls. Fluorescent SDS-PAGE gels of lysates from stable U2OS cell lines expressing defined 2-component mixtures (1-4) and 1-component controls (5-6). Cells were labeled with either JFX650-STL (S) or JFX650-HTL (H) prior to lysis, and each lane was loaded with a volume of lysate corresponding to approximately 60,000 cells. Associated figure panels are listed after each construct. 0, unbound dye.
  • FIG. 22A-22H Additional analyses of PAPA-fastSPT experiment with 2-component controls.
  • a,c,e,g A random subset of PAPA (green) and DR (violet) trajectories from individual cells. Trajectory centroids are aligned to a grid for visualization, and trajectories are displayed in order of increasing average displacement per step. Scale bar, 1 pm.
  • b,d,f,h Histograms of single-frame displacements for single-particle trajectories from all cells. Solid line, PAPA trajectories. Dashed line, DR trajectories. The sharp drop in frequency near 1 pm is due to the maximum displacement cutoff used in the particle tracking algorithm.
  • FIG. 23A-23I PAPA-SPT analysis of single-component controls. Individual proteins fused with Halo-SNAPf were labeled with a mixture of JF549-HTL and JFX650-STL, imaged with alternating green and violet photostimulation pulses, and analyzed as in FIG. 20A020F. a-c) Halo-SNAPf-3xNLS. d-f) Halo-SNAPf-H2B. g-i) Halo-SNAPf-60-mer. Note that all 60 subunits are fused to Halo-SNAPf, but only a single label is shown for clarity.
  • FIG. 25A-25F “Unmixing” of defined 2-component mixtures using PAPA.
  • Left column (a,d) Schematic of different defined mixtures of two labeled proteins, in which one protein is labeled with JFX650 only and the other is labeled with both JFX650 and JF549. Each subunit of the 60-mer is fused to SNAPf or Halo-SNAPf, though only one label is displayed for clarity.
  • PAPA was combined with single-molecule imaging to assess how self-association influences diffusion and chromatin binding by AR. Consistent with previous biochemical and live-cell imaging experiments, addition of DHT caused an increase in the overall bound fraction of AR (FIG. 26C-26D). Strikingly, PAPA trajectories had a higher bound fraction than DR trajectories, both before and after addition of DHT (FIG. 26C-26D). This supports a model in which self-association of AR increases its affinity for specific DNA sequence motifs in chromatin. Moreover, at least under the conditions of this experiment, a subset of self-associated AR molecules binds chromatin with elevated affinity even prior to addition of exogenous androgen. Thus, PAPA can be applied to monitor regulation of a biologically important proteinprotein interaction in live cells and discern its effect on chromatin binding.
  • FIG. 24A-24C PAPA analysis of androgen receptor, a) Fluorescent SDS-PAGE gel of lysates from a clonal stable U2OS cell line expressing SNAPf-mAR and Halo-mAR. Cells were stained with JFX650-STL or JFX650-HTL, and a volume of lysate corresponding to 60,000 cells was loaded per lane. MW, molecular weight in kilodaltons, b) Ensemble PAPA analysis (see FIG. 19B legend) of interaction between SNAPf-mAR and Halo-mAR. PAPA signal (green reactivation) increased after addition of DHT.
  • FIG. 26A-26D Analysis of mammalian androgen receptor using PAPA-SPT.
  • FIG. 27 Using PAPA to spotlight protein-protein interactions. 1) Label a SNAPf-tagged Target protein with a receiver fluorophore (e.g., JFX650) and a Halo-tagged Partner protein with a sender fluorophore (e.g., JF549). 2) Shelve the receiver fluorophore in the dark state using intense 633 nm illumination.
  • a receiver fluorophore e.g., JFX650
  • a Halo-tagged Partner protein e.g., JF549
  • PAPA proximity-assisted photoactivation
  • DR direct reactivation

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Abstract

The present disclosure provides methods of detecting proximity of a first molecule to a second molecule, where the first molecule comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore in a proximity-dependent manner; and where the second molecule comprises the effector fluorophore. The present disclosure also provides systems for carrying out a method of the present disclosure.

Description

PROXIMITY-DEPENDENT PHOTOACTIVATION
CROSS -REFERENCE
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/139,605, filed January 20, 2021, and U.S. Provisional Patent Application No. 63/188,290, filed May 13, 2021, which applications are incorporated herein by reference in their entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE
[0002] A Sequence Listing is provided herewith as a text file, “BERK- 439WO_SEQLIST_ST25.txt” created on December 6, 2021 and having a size of 16 KB. The contents of the text file are incorporated by reference herein in their entirety.
INTRODUCTION
[0003] A number of methods for detecting interaction of two molecules are available. Such methods include, e.g., surface plasmon resonance, fluorescence resonance energy transfer, bioluminescence resonance energy transfer, and bimolecular fluorescence complementation. However, each of these methods has its own disadvantages. There is a need in the art for effective tools and methods for studying biomolecular interactions, e.g., at single-molecule resolution in living cells.
SUMMARY
[0004] The present disclosure provides methods of detecting proximity of a first molecule to a second molecule, where the first molecule comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore in a proximity-dependent manner; and where the second molecule comprises the effector fluorophore. The present disclosure also provides systems for carrying out a method of the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1 provides a schematic depiction of an embodiment of the present disclosure.
[0006] FIG. 2A-2B depict reactivation of Janelia Fluor X 650 (JFX650), dependent on colabeling a pentameric HaloTag fusion protein with Janelia Fluor 549 (JF549).
[0007] FIG. 3A-3B depict alternating proximity-dependent and proximity-independent reactivation of Janelia Fluor 646 (JF646). [0008] FIG. 4 depicts proximity-dependent reactivation of JF646 in fixed cells.
[0009] FIG. 5 depicts reactivation of JFX650 when a HaloTag-SNAP fusion protein is doublelabeled with JFX650 SNAP ligand and JF549 HaloTag ligand.
[0010] FIG. 6A-6C depict multiple cycles of alternating illumination.
[0011] FIG. 7 depicts data showing that reactivation of JFX650 by JF549 + 561 nm light depends on proximity of the two dyes within the same molecular complex.
[0012] FIG. 8 depicts detection of inducible dimerization using proximity-dependent photoactivation.
[0013] FIG. 9 depicts detection of androgen receptor (AR) self-association using proximitydependent photoactivation.
[0014] FIG. 10 depicts a single -molecule displacement histogram from single-particle tracking (SPT) combined with proximity-dependent photoactivation. The data show that Halo- AR + SNAPf-AR oligomers, which are reactivated by 561 nm light, have a higher bound fraction.
[0015] FIG. 11A-11B depict selective highlighting of double-labeled molecules using proximity-dependent reactivation.
[0016] FIG. 12 depicts the effect of a 67-amino acid rigid alpha helical spacer on 561 nm photoactivation.
[0017] FIG. 13A-13C depict proximity-assisted photoactivation (PAPA) of JFX650 by JF549.
[0018] FIG. 14A-14D depict properties of JFX650 reactivation.
[0019] FIG. 15A-15E depict PAPA between various sender-receiver pairs.
[0020] FIG. 16A-16B depict SDS-PAGE analysis of linker constructs.
[0021] FIG. 17A-17B depict a comparison of distance-dependence of PAPA and FRET
[0022] FIG. 18A-18C depict detection of inducible dimerization using PAPA.
[0023] FIG. 19A-19B depict SDS-PAGE and PAPA traces of FRB-FKBP.
[0024] FIG. 20A-20F depict an example of “unmixing” of defined 2-component mixtures using
PAPA.
[0025] FIG. 21A-21B depict SDS-PAGE gels of defined 2-component mixtures and 1- component controls.
[0026] FIG. 22A-22H depict additional analyses of PAPA-fastSPT experiment with 2- component controls.
[0027] FIG. 23A-23I depict PAPA-SPT analysis of single-component controls.
[0028] FIG. 24A-24C depict PAPA analysis of androgen receptor.
[0029] FIG. 25A-25F depict another example of “unmixing” of defined 2-component mixtures using PAPA. [0030] FIG. 26A-26D depict analysis of mammalian androgen receptor using PAPA-SPT.
[0031] FIG. 27 schematically depicts use of PAPA to spotlight protein-protein interactions.
DEFINITIONS
[0032] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The terms also include polypeptides that have co- translational (e.g., signal peptide cleavage) and post- translational modifications of the polypeptide, such as, for example, disulfide-bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage, and the like. Furthermore, as used herein, a "polypeptide" refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art) to the native sequence, as long as the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts that produce the proteins, or errors due to polymerase chain reaction (PCR) amplification or other recombinant DNA methods.
[0033] The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule” are used interchangeably herein to include a polymeric form of nucleotides (e.g., ribonucleotides, deoxyribonucleotides, or a combination thereof). Thus, the term “polynucleotide” includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms “polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
[0034] A polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST. See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith- Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).
[0035] As used herein, the term "heterologous" used in reference to nucleic acids or polypeptides, means that these molecules are not naturally occurring in the environment (e.g., a cell) from which the heterologous nucleic acid or polypeptide was derived. As an example, a fusion polypeptide can comprise a target polypeptide and a heterologous fusion partner, where the heterologous fusion partner does not normally exist in a single polypeptide chain in nature with the target polypeptide.
[0036] By "linking" or "linker" as in "linking group," "linker moiety," etc., is meant a linking moiety that connects two groups (e.g., a rhodamine dye to a first molecule; a rhodamine dye to an affinity agent; an effector fluorophore to a second molecule; an effector fluorophore to an affinity agent; and the like) via covalent bonds. The linker may be linear, branched, cyclic or a single atom. Examples of such linking groups include alkyl, alkenylene, alkynylene, arylene, alkarylene, aralkylene, and linking moieties containing functional groups including, without limitation: amido (-NH-CO-), ureylene (-NH-CO-NH-), imide (-CO-NH-CO-) , epoxy (-O-), epithio (-S-), epidioxy (-O-O-), epidithio (-S-S-), carbonyldioxy (-O-CO-O-), alkyldioxy (-O- (CH2)n-O-), epoxyimino (-0-NH-), epimino (-NH-), carbonyl (-CO-), etc. In certain cases, one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom. The bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone. The linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group. A linker may include, without limitations, poly(ethylene glycol) unit(s) (e.g., -(CH2-CH2-O)-); amino acids; ethers; thioethers; amines; alkyls (e.g., (Ci-Ci2)alkyl), which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1- methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1 -dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3, or 4 atoms, of the cyclic group are included in the backbone. A linker may be cleavable or non-cleavable. Any convenient orientation and/or connections of the linkers to the linked groups may be used. Other examples of linking groups include oligonucleotides and polypeptides. [0037] The term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
[0038] A “host cell,” as used herein, denotes an in vivo or in vitro eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which eukaryotic or prokaryotic cells can be, or have been, used as recipients for a nucleic acid (e.g., an expression vector), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A “recombinant host cell” (also referred to as a “genetically modified host cell”) is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector. For example, a genetically modified prokaryotic host cell (e.g., a bacterium) is genetically modified by virtue of introduction into a suitable prokaryotic host cell of a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to (not normally found in nature in) the prokaryotic host cell, or a recombinant nucleic acid that is not normally found in the prokaryotic host cell. Similarly, a genetically modified eukaryotic host cell is genetically by virtue of introduction into a suitable eukaryotic host cell of a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in the eukaryotic host cell.
[0039] The term “genetic modification” refers to a permanent or transient genetic change induced in a cell following introduction of new nucleic acid (e.g., DNA exogenous to the cell) into the cell. Genetic change (“modification”) can be accomplished either by incorporation of the new nucleic acid into the genome of the host cell, or by transient or stable maintenance of the new nucleic acid as an episomal element. A permanent genetic change is generally achieved by introduction of new DNA into the genome of the cell. Permanent changes can be introduced via extrachromosomal elements such as plasmids and expression vectors, which may contain one or more selectable markers to aid in their maintenance in the recombinant host cell. Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like. The choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (i.e. in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995. [0040] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
[0041] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
[0043] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a rhodamine dye” includes a plurality of such dyes and reference to “the effector fluorophore” includes reference to one or more effector fluorophores and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
[0044] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
[0045] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
DETAILED DESCRIPTION
[0046] The present disclosure provides methods of detecting proximity of a first molecule to a second molecule, where the first molecule comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore in a proximity-dependent manner; and where the second molecule comprises the effector fluorophore. The present disclosure also provides systems for carrying out a method of the present disclosure.
METHODS FOR DETECTING PROXIMITY OF TWO MOLECULES
[0047] The present disclosure provides proximity-dependent photoactivation methods for detecting proximity of a first molecule to a second molecule, where the first molecule comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore in a proximity-dependent manner; and where the second molecule comprises the effector fluorophore. The term “effector fluorophore” is used interchangeably herein with “sender fluorophore.” The “effector fluorophore” may also include weakly fluorescent chromophores that are not typically used for fluorescence imaging.
[0048] FIG. 1 provides a schematic depiction of an embodiment of the present disclosure. A sample comprises a first molecule (“X”) that is labeled with a “receiver” fluorophore (e.g., Janelia Fluor 646 (JF646)) and a second molecule (“Y”) that is labeled with a “sender” fluorophore (e.g., Janelia Fluor 549 (JF549)) (left panel). The sample is first irradiated with light of a first wavelength (e.g., 633 nm) that inactivates JF646 and converts it to a dark state (middle panel). The sample is then irradiated with light of a second wavelength (e.g., 561 nm), thereby exciting the JF549. In some cases, excitation of JF549 causes reactivation of the adjacent darkstate JF646 fluorophore. Proximity of molecule X and molecule Y is thus determined by detecting fluorescence from JF646 upon subsequent direct excitation (e.g., with 633 nm light). As depicted in FIG. 1 , JF646 is reactivated following activation of JF549 only when molecule X and molecule Y are in sufficient proximity to one another.
[0049] Thus, the present disclosure provides a method of detecting proximity of a first molecule to a second molecule, the method comprising: a) contacting the first molecule with the second molecule to form a contacted sample, where: i) the first molecule comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore in a proximity-dependent manner; and ii) the second molecule comprises the effector fluorophore, where the contacting is carried out under conditions that inactivate the rhodamine dye; b) exciting the effector fluorophore with a second wavelength of light; and c) detecting fluorescence from the rhodamine dye in the contacted sample following excitation of the effector fluorophore. Excitation of the effector fluorophore with the second wavelength of light results in reactivation of the rhodamine dye. Fluorescence from the rhodamine dye (the reactivated rhodamine dye) indicates that the first molecule and the second molecule are in proximity to one another in the contacted sample. In some cases, fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule were in proximity to one another in the contacted sample at the time the effector fluorophore was excited. In other words, sustained contact between the first and the second molecule is not required in order for the rhodamine dye to remain reactivated. Instead, reactivation requires only that the first and second molecules be in contact at some time when the effector fluorophore is excited. For example, fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule were in proximity to one another in the contacted sample at the time the effector fluorophore was excited, even if fluorescence from the reactivated rhodamine dye is detected (and/or measured) at a time point after the effector fluorophore is excited. For example, fluorescence from the reactivated rhodamine dye can be measured at a time point that is from about 1 second (or less than 1 second) to about 1 hour (or more than 1 hour) after the effector fluorophore is excited. For example, fluorescence from the reactivated rhodamine dye can be measured at a time point that is from about 1 second to about 5 seconds, from about 5 seconds to about 10 seconds, from about 10 seconds to about 15 seconds, from about 15 seconds to about 30 seconds, from about 30 seconds to about 1 minute, from about 1 minute to about 5 minutes, from about 5 minutes to about 10 minutes, from about 10 minutes to about 15 minutes, from about 15 minutes to about 30 minutes, or from about 30 minutes to about 1 hours, or more than 1 hour, after the effector fluorophore is excited. In some cases, fluorescence from the reactivated rhodamine dye is measured within about 10 seconds of the time that the effector fluorophore is excited. For example, in some cases, fluorescence from the reactivated rhodamine dye is measured at a time point that is from about 1 millisecond to about 5 milliseconds, from about 5 milliseconds to about 10 milliseconds, from about 10 milliseconds to about 15 milliseconds, from about 15 milliseconds to about 100 milliseconds, from about 100 milliseconds to about 250 milliseconds, from about 250 milliseconds to about 500 milliseconds, or from about 500 milliseconds to about 1 second, after the effector fluorophore is excited.
[0050] In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are within 25 nm of one another. In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are within 20 nm of one another. In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are within 15 nm of one another. In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are within 10 nm of one another. For example, in some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 0.1 nm to about 25 nm from one another; e.g., detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 0.1 nm to about 0.5 nm, from about 0.5 nm to about 1 nm, from about 1 nm to about 2 nm, from about 2 nm to about
3 nm, from about 3 nm to about 4 nm, from about 4 nm to about 5 nm, from about 5 nm to about
6 nm, from about 6 nm to about 7 nm, from about 7 nm to about 8 nm, from about 8 nm to about
9 nm, from about 9 nm to about 10 nm, from about 10 nm to about 15 nm, from about 15 nm to about 20 nm, or from about 20 nm to about 25 nm, from one another. In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 0.1 nm to about 1 nm from one another. In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 0.1 nm to about 5 nm from one another. In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 0.1 nm to about 10 nm from one another. In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 0.5 nm to about 5 nm from one another. In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 1 nm to about 5 nm from one another. In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are from about 1 nm to about 10 nm from one another In some cases, detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are less than about 10 nm apart from one another; e.g., less than about 10 nm, less than about 7 nm, less than about 5 nm, less than about 3 nm, or less than about 1 nm apart from one another.
[0051] Proximity of a first molecule and a second molecule to one another can comprise binding of the first molecule to the second molecule. For example, in some cases, “proximity” of a first molecule and a second molecule to one another is due to non-covalent interaction of the first molecule with the second molecule, where non-covalent interactions can include ionic interactions, electrostatic interactions, van der Waals interactions, and the like. In other instances, “proximity” of a first molecule and a second molecule to one another is due to covalent binding of the first molecule to the second molecule.
[0052] In some cases, the proximity of the first molecule to the second molecule occurs for a period of time of less than 1 minute. For example, in some cases, the proximity of the first molecule to the second molecule occurs for a period of time of less than about 60 seconds, less than about 45 seconds, less than about 30 seconds, less than about 15 seconds, less than about 10 seconds, or less than about 5 seconds. In some cases, the proximity of the first molecule to the second molecule occurs for a period of time of from about 1 second to about 60 seconds; e.g., from about 1 second to about 5 seconds, from about 5 seconds to about 10 seconds, from about 10 seconds to about 15 seconds, from about 15 seconds to about 30 seconds, from about 30 seconds to about 45 seconds, or from about 45 seconds to about 60 seconds.
[0053] In some cases, the proximity of the first molecule to the second molecule occurs for a period of time of less than 1 second. For example, in some cases, the proximity of the first molecule to the second molecule occurs for a period of time of less than about 1 second, less than about 500 milliseconds (msec), less than about 250 msec, less than about 100 msec, less than about 50 msec, less than about 25 msec, less than about 10 msec, less than about 5 msec, or less than about 1 msec. In some cases, the proximity of the first molecule to the second molecule occurs for a period of time of from about 1 msec to about 1 second; e.g., the proximity of the first molecule to the second molecule occurs for a period of time of from about 1 msec to about 10 msec, from about 10 msec to about 25 msec, from about 25 msec to about 50 msec, from about 50 msec to about 100 msec, from about 100 msec to about 250 msec, from about 250 msec to about 500 msec, or from about 500 msec to 1 second.
[0054] In some cases, the proximity of the first molecule to the second molecule occurs for a period of time of less than 1 millisecond; for example, in some cases, the proximity of the first molecule to the second molecule occurs for a period of time of less than 1 millisecond, less than 500 nanoseconds, less than 250 nanoseconds, less than 100 nanoseconds, less than 50 nanoseconds, less than 25 nanoseconds, less than 10 nanoseconds, less than 5 nanoseconds, or about 1 nanosecond. In some cases, the proximity of the first molecule to the second molecule occurs for a period of time of from about 1 nanosecond to about 1 millisecond. For example, in some cases, the proximity of the first molecule to the second molecule occurs for a period of time of from about 1 nanosecond to about 5 nanoseconds, from about 5 nanoseconds to about 10 nanoseconds, from about 10 nanoseconds to about 25 nanoseconds, from about 25 nanoseconds to about 50 nanoseconds, from about 50 nanoseconds to about 100 nanoseconds, from about 100 nanoseconds to about 250 nanoseconds, from about 250 nanoseconds to about 500 nanoseconds, or from about 500 nanoseconds to about 1 millisecond.
[0055] In some cases, the proximity of the first molecule to the second molecule occurs for a period of time of more than 1 minute. For example, in some cases, the proximity of the first molecule to the second molecule occurs for a period of time of from about 1 minute to about 10 minutes, or more than 10 minutes.
Rhodamine dyes
[0056] As noted above, a method of the present disclosure provides for detection of proximity of a first molecule to a second molecule, where the first molecule comprises a rhodamine dye, and the second molecule comprises an effector fluorophore.
[0057] Rhodamine dyes are known in the art, and any rhodamine dye can be suitable for use in a method of the present disclosure. In some cases, a suitable rhodamine dye is a silicon- rhodamine dye. In some cases, a suitable rhodamine dye is a Janelia-Fluor, silicon-rhodamine (Si-rhodamine)-containing dye. See, e.g., Grimm et al. (2017) ACS Central Science 3:975; Grimm et al. (2015) Nature Methods 12:244; and WO 2017/201531. In some cases, a suitable rhodamine dye is JF646 (Compound 26 of Grimm et al. (2015) Nature Methods 12:244). JF646 has the following structure:
Figure imgf000013_0001
[0058] In some cases, a suitable rhodamine dye is Janelia Fluor 635 (JF635) (Zheng et al., ACS Cent. Sci. 2019, DOI: 10.1021/acscentsci.9b00676). JF635 has the following structure:
Figure imgf000014_0001
[0059] In some cases, suitable rhodamine dyes are Janelia Fluor X 646 (JFX646) or Janelia Fluor X 650 (JFX650). HaloTag ligand conjugates of JFX646 and JFX650 have the following structures (where D is deuterium):
Figure imgf000014_0002
[0060] In some cases, a suitable rhodamine dye is Janelia Fluor 585 (JF585) (Zheng et al., ACS
Cent. Sci. 2019, DOI: 10.1021/acscentsci.9b00676). JF585 has the following structure:
Figure imgf000015_0001
[0061] In some cases, a suitable rhodamine dye is Janelia Fluor 608 (JF608) (Zheng et al., ACS Cent. Sci. 2019, DOI: 10.1021/acscentsci.9b00676). JF608 has the following structure:
Figure imgf000015_0002
[0062] In some cases, a suitable rhodamine dye is Alexa Fluor 633. Alexa Fluor 633 has the following structure:
Figure imgf000015_0003
Effector fluorophores
[0063] Fluorophores (e.g., fluorescent dyes; fluorescent proteins) are molecules (e.g., dyes; polypeptides) that, when irradiated with light of a wavelength (a first wavelength) which they absorb, can emit light of a different wavelength (a second wavelength). Suitable effector fluorophores include, e.g., a rhodamine dye; a fluorescent polypeptide; and the like. As noted above, the term “effector fluorophore” is used interchangeably herein with “sender fluorophore.”
[0064] A suitable effector fluorophore emits light such that the inactivated rhodamine dye becomes reactivated. The effector fluorophore need not emit light of a wavelength that would directly reactivate the inactivated rhodamine dye. For example, a suitable effector fluorophore is JF549, which emits at 590 nm. A second molecule comprising JF549 can, when in sufficient proximity to a first molecule comprising JF646 that has been inactivated by exposure to light of 633 nm, reactivate the inactivated JF646, even though JF646 is not reactivated by exposure to 590 nm light that is emitted by JF549. Thus, a suitable effector fluorophore is one that reactivates the inactivated rhodamine dye (that is linked to the first molecule) in a proximitydependent manner; i.e., the effector fluorophore is linked to the second molecule (directly or via a linker) and reactivates that inactivated rhodamine dye (that is linked to the first molecule) only when the first molecule and the second molecule are in sufficiently close proximity to one another (as described above).
[0065] Suitable effector fluorophores include, but are not limited to, JF549, Alexa Fluor 532, Alexa Fluor 549, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, tetramethylrhodamine, ATTO 532, ATTO Rho6G, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol l, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO590, ATTO Rhol3, or ATTO594.
[0066] As another example, a suitable effector (“sender”) fluorophore is Janelia fluorophore 526 (JF526). The structure of JF526 is as follows:
Figure imgf000017_0001
[0067] In some cases, the effector fluorophore is a fluorescent polypeptide such as mKO, mK02, DsRed, Turbo red fluorescent protein (RFP), TagRFP, tdTomato, mOrange, or O- GECO1.
[0068] As an example, a DsRed polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
MRSSKNVIKE FMRFKVRMEG TVNGHEFEIE GEGEGRPYEG HNTVKLKVTK GGPLPF AWDI LSPQFQYGSK VYVKHPADIP DYKKLSFPEG FKWERVMNFE DGGVVTVTQD SS LQDGCFIY KVKFIGVNFP SDGPVMQKKT MGWEASTERL YPRDGVLKGE IHKALKLK DG GHYLVEFKSI YMAKKPVQLP GYYYVDSKLD ITSHNEDYTI VEQYERTEGR HHLF (SEQ ID NO:1).
[0069] As another example, an mK02 polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
MVSVIKPEMK MRYYMDGSVN GHEFTIEGEG TGRPYEGHQE MTLRVTMAEG GPMPF AFDLV SHVFCYGHRV FTKYPEEIPD YFKQAFPEGL SWERSLEFED GGSASVSAHI SLR GNTFYHK SKFTGVNFPA DGPIMQNQSV DWEPSTEKIT ASDGVLKGDV TMYLKLEGG G NHKCQMKTTY KAAKEILEMP GDHYIGHRLV RKTEGNITEQ VEDAVAHS (SEQ ID NO:2). [0070] In some cases, the effector fluorophore is JF549; see, e.g., Grimm et al. (2015) Nature Methods 12:244.
[0071] JF549 has the following structure:
Figure imgf000018_0001
[0072] In some cases, the effector fluorophore is JFX549. The structure of JFX549 is as follows:
Figure imgf000018_0002
[0073] In some cases, the effector fluorophore is not a cyanine dye such as Cy3 or Cy5. Molecules
[0074] The first molecule and the second molecule can be any of a variety of molecules, including synthetic (non-naturally-occurring) molecules, biomolecules, nucleotides, nucleotide analogs, sugars, sugar analogs, small molecules (e.g., small molecule drugs), and the like. Suitable molecules (which may be biomolecules) include, but are not limited to, polypeptides, nucleic acids, lipids, sugars (e.g., mannose, lactose, glucose, galactose, N-acetyl-glucosamine, N-acetyl-galactosamine, fucose, and the like), polysaccharides, and molecules comprising one or more of the foregoing. For example, a molecule can be a lipopolysaccharide, a glycoprotein, a glycolipid, a lipoprotein, a proteoglycan, and the like. Suitable biomolecules include, e.g., RNA (e.g., mRNA, gRNA, miRNA, piRNA, sgRNA, shRNA, siRNA, stRNA, sncRNA, tncRNA, snoRNA, smRNA, shRNA, snRNA, tRNA, tracrRNA, or other small non-coding RNA), DNA, peptide nucleic acids, combinations of RNA and DNA (e.g., chimeraplasts), and the like.
[0075] In some cases, the first molecule and the second molecule are in vitro outside of a cell (e.g., in an acellular environment). In some cases, the first molecule and the second molecule are present in a living cell in vitro. In some cases, the first molecule and the second molecule are present in a multicellular organism; e.g., in some cases, the first molecule and the second molecule are in vivo. Where the first molecule and the second molecule are in vivo, in some cases, the first molecule and the second molecule are inside of a cell. Where the first molecule and the second molecule are in vivo, in some cases, the first molecule and the second molecule are outside of a cell; e.g., the first molecule and the second molecule are in extracellular fluid. In some cases, the first molecule is present on the surface of a cell; and the second molecule is outside of the cell. In some cases, the first molecule and the second molecule are inside a living cell within a cultured organoid or tissue explant. In some cases, the first molecule and the second molecule are in the same organelle within a eukaryotic cell. In some cases, the first molecule and the second molecule are in different organelles within a eukaryotic cell.
[0076] Suitable polypeptide biomolecules include, e.g., receptors, immunomodulatory polypeptides, cytokines, chemokines, peptide hormones, differentiation factors, growth factors, enzymes, antibodies, peptide neurotransmitters, transcriptional repressors, transcriptional activators, nucleic acid binding polypeptides (e.g., chromatin and the like), structural polypeptides, and the like. Suitable polypeptide biomolecules include structural proteins; receptors; enzymes; cell surface proteins; proteins integral to the function of a cell; proteins involved in catalytic activity; proteins involved in motor activity; proteins involved in helicase activity; proteins involved in metabolic processes (anabolism and catabolism); proteins involved in antioxidant activity; proteins involved in proteolysis; proteins involved in biosynthesis; proteins having kinase activity; proteins having oxidoreductase activity; proteins having transferase activity; proteins having hydrolase activity; proteins having lyase activity; proteins having isomerase activity; proteins having ligase activity; proteins having enzyme regulator activity; proteins having signal transducer activity; structural polypeptides; polypeptides having binding activity; receptor polypeptides; proteins involved in cell motility; proteins involved in membrane fusion; proteins involved in cell communication; proteins involved in regulation of biological processes; proteins involved in development; proteins involved in cell differentiation; proteins involved in response to stimulus; behavioral proteins; cell adhesion proteins; proteins involved in cell death; proteins involved in transport (including protein transporter activity, nuclear transport, ion transporter activity, channel transporter activity, and the like); proteins involved in secretion activity; proteins involved in electron transporter activity; proteins involved in pathogenesis; proteins involved in chaperone regulator activity; proteins having nucleic acid binding activity; proteins having transcription regulator activity; proteins involved in extracellular organization; proteins involved in biogenesis; proteins involved in translation regulation; and the like.
[0077] Suitable polypeptides can be of any of a variety of origins, and can thus include mammalian polypeptides, insect polypeptides, arachnid polypeptides, invertebrate polypeptides, viral polypeptides, bacterial polypeptides, archaeal polypeptides, and the like.
[0078] Suitable biomolecules include nucleic acids. In some cases, the nucleic acid is a DNA molecule. In some cases, the nucleic acid is an RNA molecule. In some cases, the nucleic acid comprises both deoxyribonucleotides and ribonucleotides. In some cases, the nucleic acid is a single-stranded DNA molecule. In some cases, the nucleic acid is a double-stranded DNA molecule. In some cases, the nucleic acid is a single-stranded RNA molecule. Suitable nucleic acids include, e.g., a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a ribozyme, and the like. Suitable nucleic acids include nucleic acids that are or act as siRNAs or other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, self-cleaving RNAs, ribozymes, fragment thereof and/or variants thereof (such as Peptidyl transferase 23S rRNA, RNase P, Group I and Group II introns, GIRI branching ribozymes, Leadzyme, Hairpin ribozymes, Hammerhead ribozymes, HDV ribozymes, Mammalian CPEB3 ribozyme, VS ribozymes, glmS ribozymes, CoTC ribozyme, etc.), microRNAs, microRNA mimics, supermirs, aptamers, antimirs, antagomirs, U1 adaptors, triplex-forming oligonucleotides, RNA activators, long non-coding RNAs, short non-coding RNAs (e.g., piRNAs), immunomodulatory oligonucleotides (such as immunostimulatory oligonucleotides, immunoinhibitory oligonucleotides), GNA, LNA, ENA, PNA, TNA, HNA, TNA, XNA, HeNA, CeNA, morpholinos, G-quadruplex (RNA and DNA), antiviral oligonucleotides, and decoy oligonucleotides. [0079] Suitable lipids include, e.g., 3 -N- [(methoxypoly (ethylene glycol) 2000) carbamoyl] -1,2- dimyristyloxy-propylamine (PEG-C-DMA), 1 ,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, dipalmitoylphosphatidylcholine, 3-N-[(w-methoxy poly(ethylene glycol)2000)carbamoyl]-l,2- dimyrestyloxypropylamine, 1 ,2-dilinoleyloxy-3-N,Ndimethylaminopropane, 1 ,2-distearoyl-sn- glycero-3-phosphocholine, PEG-cDMA, 1 ,2-dilinoleyloxy-3-(N ;N-dimethyl)aminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), and the like.
[0080] Suitable molecules include small molecules such as drugs, e.g., where a small molecule can have a molecular weight in the range of from about 5 Daltons to 2500 Daltons. Small molecules may comprise functional groups necessary for structural interaction with other molecules such as proteins, where such interactions may be via hydrogen bonding. Small molecules can include one or more of an amine, a carbonyl, a hydroxyl, or a carboxyl group. Small molecules may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Small molecule can also include amino acids, sugars, oligosaccharides, fatty acids, steroids, purines, or pyrimidines, as well as derivatives, structural analogs or combinations thereof. Suitable small molecules include, e.g., vitamins, neurotransmitters, ligands for receptors, monosaccharides, disaccharides, and the like. Suitable small molecules include therapeutic agents, e.g., cancer chemotherapeutic agents, anti-inflammatory agents, anti-hypertensive agents, and the like.
[0081] In some cases, the first molecule or the second molecule is a synthetic polymer. Suitable synthetic polymers include, but are not limited to, polyalkylenes such as polyethylene and polypropylene and polyethyleneglycol (PEG); poly chloroprene; polyvinyl ethers such as poly(vinyl acetate); polyvinyl halides such as poly (vinyl chloride); poly siloxanes; polystyrenes; polyurethanes; polyacrylates such as poly(methyl (meth)acrylate), poly(ethyl (meth) acrylate), poly(n-butyl(meth)acrylate), poly(isobutyl (meth) acrylate), poly(tert-butyl (meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate); polyacrylamides such as poly( acrylamide), poly (methacrylamide), poly(ethyl acrylamide), poly (ethyl methacrylamide), poly(N-isopropyl acrylamide), poly(n, iso, and tert-butyl acrylamide); and copolymers and mixtures thereof.
[0082] In some cases, the first molecule comprises a first polypeptide; and the second molecule comprises a second polypeptide. In some cases, the first molecule comprises a nucleic acid; and the second molecule comprises a second nucleic acid. In some cases, the first molecule comprises a nucleic acid; and the second molecule comprises a polypeptide. In some cases, the first molecule comprises a polypeptide; and the second molecule comprises a nucleic acid. In some cases, the first molecule comprises a lipid; and the second molecule comprises a polypeptide. In some cases, the first molecule comprises a polypeptide; and the second molecule comprises a lipid. In some cases, the first molecule comprises a polysaccharide; and the second molecule comprises a polypeptide. In some cases, the first molecule comprises a polypeptide; and the second molecule comprises a polysaccharide.
Heterologous moieties
[0083] The first molecule and/or the second molecule can include a heterologous moiety such as an affinity tag, a self-labelling polypeptide, and the like.
[0084] The first molecule and/or the second molecule can include, as a heterologous moiety, an affinity tag. Suitable affinity tags include, for example, a histidine tag (His tag), a chitin-binding domain, a calmodulin tag, a polyglutamate tag, a maltose binding protein, glutathione-S- transferase, an S-tag, an SBP-tag (Keefe et al. (2001) Protein Expr. Purif. 23:440), a Strep-tag, Strep-tag II (e.g., an 8-amino acid peptide of the amino acid sequence Trp-Ser-His-Pro-Gln-Phe- Glu-Lys), a fluorescent protein, a thioredoxin tag, a Nus-tag, an immunoglobulin Fc tag, a HALO tag, a FLAG tag, a V5 tag, a VSV tag, an Xpress tag, an E tag, a Myc-tag, a hemagglutinin (HA) tag, a Softag, an NE tag, biotin, BirA, AviTag, BCCP, SpyTag (Zakeri et al. (2012) Proc. Natl. Acad. Sci. USA 109:E690; Anuar et al. (2019) Nature Communications 10:1734), SpyCatcher (e.g., a peptide of the sequence: AHIVMDAYKPTK (SEQ ID NO:3)), SnoopTag, and SnoopCatcher (e.g., a peptide of the sequence: KLGDIEFIKVNK (SEQ ID NO:4)). See, e.g., Hatlem et al. (2019) Int. J. Mol. Sci. 20:2129.
[0085] The first molecule and/or the second molecule can include, as a heterologous moiety, a self-labelling polypeptide. Suitable self-labelling polypeptides (also referred to herein as “selflabelling protein tags”) include a SNAP polypeptide, a CLIP polypeptide, or a HALO polypeptide. Also suitable for use is a halo-based oligonucleotide binder (HOB) polypeptide. See, e.g., Kossman et al. (2016) Chembiochem. 17:1102. A HOB polypeptide binds chlorohexyl moieties. Also suitable for use is a trimethoprim (TMP) tag, an engineered form of E. coli dihydrofolate reductase (DHFR) that forms a non-covalent high-affinity complex with trimethoprim derivatives. See, e.g., Gallagher et al. (2009) ACS Chem. Biol. 4:547; and Jing and Cornish (2013) ACS' Chem. Biol. 8:1704.
[0086] A SNAP polypeptide can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEVPAPAAVLGGPEPL MQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYSHLA ALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLAVKEWLLAHEGHRL GKPGLG (SEQ ID NO:5). A SNAP polypeptide binds O6-benzylguanine (BG).
[0087] A CLIP polypeptide can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEVPAPAAVLGGPEPL IQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISESHLA ALVGNPAATAAVNTALDGNPVPILIPCHRVVQGDSDVGPYLGGLAVKEWLLAHEGHRL GKPGLG (SEQ ID NO:6). A CLIP polypeptide can bind O2 -benzylcytosine (BC).
[0088] A HALO polypeptide can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTH RCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAK RNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVV RPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKL LFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISG (SEQ ID NO:7). A HALO polypeptide binds chloroalkane.
[0089] The first molecule and/or the second molecule can include, as a heterologous moiety, a sortase. Suitable sortases include sortase A (SrtA), sortase B (SrtB), sortase C (SrtC), sortase D (SrtD), sortase E (SrtE), sortase F (SrtF), and variants thereof.
Labeling of molecules
[0090] As noted above, a method of the present disclosure provides for detection of proximity of a first molecule to a second molecule, where the first molecule comprises a rhodamine dye, and the second molecule comprises an effector fluorophore. The rhodamine dye can be linked to the first molecule directly or via a linker. Similarly, effector fluorophore can be linked to the second molecule directly or via a linker.
[0091] In some cases, a rhodamine dye is linked to the first molecule, or an effector fluorophore is linked to the second molecule, via a HALO tag, a SNAP tag, a CLIP tag, a sortase polypeptide, dihydrofolate reductase (DHFR), tetracysteine, and the like. [0092] In some cases, rhodamine dye or the effector fluorophore is linked to an affinity agent such as a benzylnucleoside base or a chloropyrimidine. In some cases, rhodamine dye or the effector fluorophore is linked to a benzylnucleoside base, such as benzylguanine or benzylcytosine. In some cases, the rhodamine dye or the effector fluorophore is linked to benzylguanine. In embodiments where the rhodamine dye or the effector fluorophore is linked to benzylguanine, the benzylguanine can provide for covalent binding to a SNAP tag, e.g., where the first molecule or the second molecule comprises the SNAP tag. In some cases, the rhodamine dye or the effector fluorophore is linked to benzylcytosine. In embodiments where the rhodamine dye or the effector fluorophore is linked to benzylcytosine, the benzylcytosine can provide for covalent binding to a CLIP tag, e.g., where the first molecule or the second molecule comprises the CLIP tag. In some cases, the affinity agent is a chloropyrimidine; a chloropyrimidine can bind to a SNAP tag. In some cases, the affinity agent is O6-benzylguanine (e.g., a substrate for SNAP). In some cases, the affinity agent is O6-(5-pyridylmethyl)guanine (e.g. a substrate for SNAP).
[0093] Suitable affinity agents also include alkyl derivatives, such as haloalkyl derivatives where one or more hydrogen atoms in an alkyl or alkyl derivative is replaced by a halogen, e.g., fluoro, chloro, or bromo. In some cases, the haloalkyl derivative is a fluoroalkane. In some cases, the haloalkyl derivative is a chloroalkane. In some cases, the haloalkyl derivative is a bromoalkane. In some cases, the affinity agent is chloroalkane, such as CKCEL OCELCEL^. In embodiments where the affinity agent is chloroalkane, the chloroalkane affinity agent can provide for covalent binding to a HALO tag.
[0094] In some cases, a rhodamine dye is attached to the first molecule and/or the effector fluorophore is attached to the second molecule via a linker. A linker is any suitable moiety capable of linking, connecting, or tethering a rhodamine dye to a first molecule or an effector fluorophore to a second molecule. A linker can be a polymer of one or more repeating or nonrepeating monomer units (e.g., nucleic acid, amino acid, carbon-containing polymer, carbon chain, etc.). In some cases, a linker is a peptide linker. A linker can comprise any chemical moiety with a functional (or reactive) group that can be linked to a rhodamine dye (or a moiety linked to a rhodamine dye) or an effector fluorophore (or a moiety linked to an effector fluorophore). Any suitable moiety capable of tethering the signal and interaction elements may find use as a linker.
[0095] A variety of linker groups can be used. Suitable linkers can comprise, e.g., an alkyl group, a methylene carbon chain, an ether, a polyether, an alkyl amide linker, a peptide linker, a modified peptide linker, a poly(ethylene glycol) (PEG) linker, a streptavidin-biotin or avidinbiotin linker, polyaminoacids (e.g. polylysine), functionalized PEG, polysaccharides, glycosaminoglycans, dendritic polymers an oligonucleotide linker, a phospholipid linker, an alkenyl chain, an alkynyl chain, a disulfide, or a combination thereof. In some cases, the linker is cleavable.
[0096] Peptide linkers include, e.g., peptides from about 4 amino acids to about 300 amino acids in length. For example, suitable peptide linkers can be from about 4 amino acids to about 25 amino acids in length, from about 25 amino acids to about 50 amino acids in length, from about 50 amino acids to about 100 amino acids in length, from about 100 amino acids to about 200 amino acids in length, or from about 200 amino acids to about 300 amino acids in length. Suitable peptide linkers include glycine-serine polymers such as (GGGGS)n (SEQ ID NO:8), (GSGGS)n (SEQ ID NO:9), (GGGS)n (SEQ ID NO: 10), and the like, where n is an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Suitable peptide linkers include, e.g., (GGGGS)n (SEQ ID NO:8), where n is 1, 2, or 3. Suitable peptide linkers also include longer (up to about 300 amino acids) artificial low-complexity sequences that are designed to be flexible, non-self- interacting, and solvent-exposed. Suitable peptide linkers include flexible polypeptide sequences from naturally occurring proteins.
[0097] In some cases, the conformational flexibility of the linker(s) will allow the rhodamine dye and the effector fluorophore to come in contact repeatedly during a photoactivation pulse. Varying the linker length and activation pulse duration can also provide a means of gauging the spatial distance between the first molecule and the second molecule, and of extending the distance range of detection. Suitable linkers include peptide linkers (e.g., flexible peptide linkers), oligonucleotides, and synthetic polymers.
[0098] A rhodamine dye can be linked to a first molecule using any of a variety of standard chemistries. Suitable chemistries include, e.g., maleimide/thiol; thiol/thiol; pyridyldithiol/thiol; succinimidyl iodoacetate/thiol; N-succinimidylester (NHS ester), sulfodicholorphenol ester (SDP ester), or pentafluorophenyl-ester (PFP ester)/amine; bissuccinimidylester/amine; imidoesters/amines; hydrazine or amine/aldehyde, dialdehyde or benzaldehyde; isocyanate/hydroxyl or amine; carbohydrate-periodate/hy dr azine or amine; diazirine/aryl azide chemistry; pyridyldithiol/aryl azide chemistry; alkyne/azide; carboxy-carbodiimide/amine; amine/Sulfo-SMCC (Sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane- 1 - carboxy late)/thiol; and amine/BMPH (N-[-Maleimidopropionic acid] hydrazide TFA)/thiol. Suitable chemistries also include click chemistry. Similarly, an effector fluorophore can be linked to a second molecule using any of a variety of standard chemistries, including any of the aforementioned chemistries. [0099] Proteins may be labeled with rhodamine dyes or effector dyes by incorporation of unnatural amino acids (reviewed by Elia et al., 2020, The FEBS Journal, DOI: 10.1111/febs.l5477).
[00100] For labeling of a nucleic acid, a conjugate of an effector fluorophore or a rhodamine dye to a DNA-binding dye can be used. DNA-binding dyes are known in the art. DNA-binding dyes include, but are not limited to, DAPI (4’,6-diamidino-2-phenylindole), 7-AAD (7- aminoactinomycin D), Hoechst 33342, Hoechst 33258, Hoechst 34580, intercalators comprising a lanthanide chelate, and cyanine dyes such as SYBR Green™ and PicoGreen™.
[00101] As one non-limiting example, Hoechst 33342, a double-stranded DNA-binding dye, can be conjugated to JF549 to label DNA with JF549. The DNA-Hoechst 33342 -JF549 conjugate (JF549-labelled second molecule) can be used to selectively photoactivate a JF646-labeled molecule that binds to the DNA. (Legant et al., 2016, Nature Methods, DOI: 10.1038/nmeth.3797).
[00102] For labeling of membranes (e.g., biological membranes), a conjugate of an effector fluorophore or a rhodamine dye to a lipid such as cholesterol can be used. For example, JF549 can be conjugated to cholesterol (or other membrane lipid); and the JF-549-lipid conjugate can be used to selectively photoactivate a JF646-labeled molecule that is associated with a membrane.
Detection of fluorescence
[00103] As noted above, a subject method comprises detecting fluorescence from the reactivated rhodamine dye in the contacted sample. Detection of fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule were within sufficient proximity of one another (e.g., from about 0.1 nm to about 25 nm from one another, as described above) in the contacted sample when the effector fluorophore was excited. Fluorescence from the reactivated rhodamine dye can be detected using any of a variety of known techniques and instrumentation. For example, fluorescence can be detected using a fluorescence microscope, a fluorometer, a fluorescence scanner, or a flow cytometer.
Controls
[00104] In some cases, a subject method further comprises normalization of fluorescence, e.g., by exposing the sample (comprising the first molecule comprising the inactivated rhodamine dye and the second molecule comprising the effector fluorophore) to light of a wavelength that directly reactivates the inactivated rhodamine dye independent of proximity to the effector dye. Such proximity-independent reactivation of the inactivated rhodamine dye can serve as a quantitative internal standard to which proximity-dependent reactivation of the inactivated rhodamine dye may be normalized.
[00105] As an example, where the first molecule comprises JF646 as the rhodamine dye, the JF646 can be inactivated by exposure to light of 633 nm, thereby generating a sample (an “inactivated sample”) comprising a first molecule comprising inactivated JF646. Exposure of the inactivated sample to light of a wavelength of less than 500 nm (e.g., from about 300 nm to about 400 nm, from about 400 nm to about 495 nm, from about 400 nm to about 450 nm, or from about 405 nm to about 488 nm) can reactivate the JF646 in a proximity-independent manner. In some cases, exposure of the inactivated sample to light of a wavelength of from about 405 nm to about 488 nm reactivates the JF646 in a proximity-independent manner. In some cases, exposure of the inactivated sample to light of a wavelength of about 488 nm reactivates the JF646 in a proximity-independent manner.
[00106] In contrast, as discussed above, a suitable effector fluorophore is one that reactivates the inactivated rhodamine dye (that is linked to the first molecule) in a proximity-dependent manner; i.e., the effector fluorophore is linked to the second molecule (directly or via a linker) and reactivates that inactivated rhodamine dye (that is linked to the first molecule) only when the first molecule and the second molecule are in sufficiently close proximity to one another (as described above). For example, a suitable effector fluorophore is JF549, which emits at a range of wavelengths including 561 nm and 590 nm. A second molecule comprising JF549 can, when in sufficient proximity to a first molecule comprising JF646 that has been inactivated by exposure to light of 633 nm, reactivate the inactivated JF646, even though JF646 is not directly reactivated by exposure to light of either 561 nm or 590 nm.
[00107] In some cases, an inactivated sample (a sample comprising: i) a first molecule comprising a rhodamine dye that has been inactivated by exposure to light of an inactivating wavelength; and ii) a second molecule comprising an effector fluorophore) is exposed to: a) light of a wavelength that excites the effector fluorophore, such that the effector fluorophore reactivates the inactivated rhodamine dye in a proximity-dependent manner; and b) light of a wavelength that directly reactivates the inactivated rhodamine dye in a proximity-independent manner. The exposure to the (a) light of a wavelength that excites the effector fluorophore, such that the effector fluorophore reactivates the inactivated rhodamine dye in a proximity-dependent manner; and (b) light of a wavelength that directly reactivates the inactivated rhodamine dye in a proximity-independent manner can be carried out simultaneously. The exposure to the (a) light of a wavelength that excites the effector fluorophore, such that the effector fluorophore reactivates the inactivated rhodamine dye in a proximity-dependent manner; and (b) light of a wavelength that directly reactivates the inactivated rhodamine dye in a proximity-independent manner can be carried out at different times, e.g., in an alternating manner.
[00108] The ratio of the fluorescence from proximity-dependent reactivation of the inactivated rhodamine dye to the fluorescence from proximity-independent reactivation of the inactivated rhodamine dye can indicate what fraction of molecules labeled with the rhodamine dye are in proximity to molecules labeled with the effector fluorophore. Alternatively, the difference in proximity-dependent and proximity-independent reactivation can be used as a measure of proximity.
UTILITY
[00109] A proximity-dependent photoactivation method of the present disclosure is useful for a variety of research and diagnostic applications. For example, a method of the present disclosure can be used for live cell single-molecule imaging, super-resolution imaging, and related imaging-based methods.
[00110] As an example, a proximity-dependent photoactivation method of the present disclosure can be used to detect binding of two molecules (a first molecule and a second molecule; such as two polypeptides, a polypeptide and a polynucleotide, etc., as described above) in a living cell in vitro. The effect of various compounds and/or conditions on the binding of the two molecules to one another can be determined using a proximity-dependent photoactivation method of the present disclosure. For example, binding of a first molecule to a second molecule can be carried out in the presence of a test compound (e.g., a small molecule (e.g., a drug), a cytokine, a chemokine, a ligand for a cell-surface receptor, and the like); and the effect of the test compound on binding of the first molecule to the second molecule can be determined by comparing the binding in the presence of the test compound to the binding in a control sample which does not include the test compound (i.e., binding in the absence of the test compound).
[00111] Similarly, the effect of a condition on the binding of a first molecule to a second molecule can be tested by determining the effect of the condition on the binding, and comparing the binding in the presence of the condition to the binding in the absence of the condition. Such conditions can include, e.g., pH, temperature, osmolarity, contacting the cell comprising the first and second molecules to a second cell (cell-cell contact), and the like.
[00112] As another example, association of a protein with a cell membrane can be identified using a proximity-dependent photoactivation method of the present disclosure. For labeling of membranes (e.g., biological membranes), a conjugate of an effector fluorophore or a rhodamine dye to a lipid such as cholesterol can be used. For example, a cell membrane can be labeled by incorporation into the cell membrane of an effector fluorophore-labeled membrane lipid. The effector fluorophore-lipid conjugate integrates into the cell membrane, and can reactivate an inactivated rhodamine dye that is conjugated to a first molecule when the first molecule binds to the membrane. For example, JF549 can be conjugated to cholesterol (or other membrane lipid); and the JF549-lipid conjugate can be incorporated into a cell membrane, where it can selectively photoactivate a JF646-labeled molecule (e.g., a JF646-labeled protein) that is associated with a membrane.
[00113] As another example, labeling of a small molecule drug with an effector fluorophore can be used to selectively photoactivate a rhodamine-dye-labeled protein, where the rhodamine dye has been inactivated, to determine binding of the drug to the protein.
[00114] As another example, a proximity-dependent photoactivation method of the present disclosure can be used to detect post-translational modification of a protein with a modifying group by labeling the modifying group with an effector fluorophore and the protein with a rhodamine dye. For example, an effector fluorophore -labeled sugar can be used to detect post- translational glycosylation of a rhodamine dye-labeled protein. As another example, an effector fluorophore-labeled HALO tag-ubiquitin fusion protein could be used to detect ubiquitinylation of rhodamine dye-labeled protein.
[00115] A proximity-dependent photoactivation method of the present disclosure can be used in combination with a super-resolution imaging method to reveal both three-dimensional structure and molecular interactions within cells.
[00116] As another example, a proximity-dependent photoactivation method of the present disclosure can be used to detect aggregation of labeled proteins, including small oligomeric aggregates that are not clearly visible by other means. Small oligomers may contribute to the pathological effects of aggregation-prone proteins (such as amyloid beta in Alzheimer's disease) and presage the formation of larger aggregates.
SYSTEMS
[00117] The present disclosure provides systems for carrying out a proximity-dependent photoactivation method of the present disclosure. A system of the present disclosure can comprise: a) a first molecule that comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore; b) a second molecule that comprises the effector fluorophore; and c) a light source that is configured to produce the first wavelength of light that inactivates a rhodamine dye. In some cases, the light source is also configured to produce a wavelength of light that excites the effector fluorophore. Examples of Non-Limiting Aspects of the Disclosure
[00118] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:
[00119] Aspect 1. A method of detecting proximity of a first molecule to a second molecule, the method comprising: a) contacting the first molecule with the second molecule to form a contacted sample, wherein: i) the first molecule comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore in a proximity-dependent manner; and ii) the second molecule comprises the effector fluorophore, and wherein said contacting is carried out under conditions that inactivate the rhodamine dye; b) exciting the effector fluorophore with a second wavelength of light, wherein the excited effector fluorophore reactivates the inactivated rhodamine dye; and c) detecting fluorescence from the reactivated rhodamine dye in the contacted sample, wherein fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are within 25 nm, within 20 nm, within 15 nm, or within 10 nm of one another in the contacted sample.
[00120] Aspect 2. The method of aspect 1, wherein the rhodamine dye is inactivated at 633 nm.
[00121] Aspect 3. The method of aspect 1 or aspect 2, wherein the rhodamine dye is a silicon rhodamine dye, optionally wherein the rhodamine dye is Janelia Fluor 646, Janelia Fluor X 646 or Janelia Fluor X 650.
[00122] Aspect 4. The method of aspect 1 , wherein the effector fluorophore is a second rhodamine dye or a fluorescent polypeptide.
[00123] Aspect 5. The method of aspect 4, wherein the effector fluorophore is JF549, JFX549, JF526, Alexa Fluor 532, Alexa Fluor 549, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, tetramethylrhodamine, ATTO 532, ATTO Rho6G, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol l, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO590, ATTO Rhol3, or ATTO594.
[00124] Aspect 6. The method of any one of aspects 1-5, wherein the first molecule is a biomolecule. [00125] Aspect 7. The method of any one of aspects 1-6, wherein the second molecule is a biomolecule.
[00126] Aspect 8. The method of aspect 6 or aspect 7, wherein:
[00127] a) the first biomolecule is selected from the group consisting of a nucleic acid, a lipid, a carbohydrate, a small molecule, and a polypeptide; and
[00128] b) the second biomolecule is selected from the group consisting of a nucleic acid, a lipid, a carbohydrate, a small molecule, and a polypeptide.
[00129] Aspect 9. The method of any one of aspects 6-8, wherein the first biomolecule is a polypeptide and wherein the second biomolecule is a polypeptide.
[00130] Aspect 10. The method of any one of aspects 6-8, wherein:
[00131] i) the first biomolecule is a nucleic acid and wherein the second biomolecule is a polypeptide; or
[00132] ii) the first biomolecule is a polypeptide and the second biomolecule is a nucleic acid; or
[00133] iii) the first biomolecule is a nucleic acid and the second biomolecule is a nucleic acid.
[00134] Aspect 11. The method of any one of aspects 6-8, wherein:
[00135] i) the first biomolecule is a lipid and wherein the second biomolecule is a polypeptide; or
[00136] ii) the first biomolecule is a polypeptide and the second biomolecule is a lipid.
[00137] Aspect 12. The method of any one of aspects 6-8, wherein:
[00138] i) the first biomolecule is a carbohydrate and wherein the second biomolecule is a polypeptide; or
[00139] ii) the first biomolecule is a polypeptide and the second biomolecule is a carbohydrate.
[00140] Aspect 13. The method of any one of aspects 1-10, wherein the first molecule is a fusion polypeptide that comprises a heterologous fusion polypeptide selected from a SNAP, a CLIP, a HALO polypeptide, a dihydrofolate reductase (DHFR) polypeptide, a tetracysteine tag, a sortase polypeptide, or a transglutaminase polypeptide, and wherein the rhodamine dye is linked to the first molecule via the SNAP, CLIP, HALO polypeptide, DHFR polypeptide, tetracysteine tag, sortase polypeptide, or transglutaminase polypeptide.
[00141] Aspect 14. The method of any one of aspects 1-10, wherein the second molecule is a fusion polypeptide that comprises a heterologous fusion polypeptide selected from a SNAP, a CLIP, or a HALO polypeptide, and wherein the effector fluorophore is linked to the first molecule via the SNAP, CLIP, or HALO polypeptide.
[00142] Aspect 15. The method of any one of aspects 1-14, wherein the first molecule and the second molecule are in vitro outside of a cell. [00143] Aspect 16. The method of any one of aspects 1-14, wherein the first molecule and the second molecule are present in a cell in vitro.
[00144] Aspect 17. The method of any one of aspects 1-14, wherein the first molecule and the second molecule are present in a cell in vivo.
[00145] Aspect 18. The method of aspect 16 or aspect 17, wherein the cell is a eukaryotic cell.
[00146] Aspect 19. The method of aspect 18, wherein the cell is present in a tissue or an organ.
[00147] Aspect 20. The method of any one of aspects 1-19, wherein said detecting is carried out using a microscope.
[00148] Aspect 21. The method of any one of aspects 1-20, further comprising reactivating the inactivated rhodamine dye by exposing the contacted sample to light of a wavelength of less than 500 nm, thereby activating the rhodamine dye in a proximity-independent manner.
[00149] Aspect 22. The method of aspect 21, comprising reactivating the inactivated rhodamine dye by exposing the contacted sample to light of a wavelength of from about 405 nm to about 488 nm.
[00150] Aspect 23. A system comprising:
[00151] a) a first molecule that comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore;
[00152] b) a second molecule that comprises the effector fluorophore; and
[00153] c) a light source that is configured to produce the first wavelength of light that inactivates a rhodamine dye.
[00154] Aspect 24. The system of aspect 23, wherein the light source is configured to produce a wavelength of light that excites the effector fluorophore.
[00155] Aspect 25. The system of aspect 23 or aspect 24, wherein:
[00156] a) the rhodamine dye is a silicon rhodamine dye, optionally wherein the rhodamine dye is Janelia Fluor 646, Janelia Fluor X 646 or Janelia Fluor X 650; and
[00157] b) the effector fluorophore is a second rhodamine dye or a fluorescent polypeptide, optionally wherein the effector fluorophore is JF549, JFX549, JF526, Alexa Fluor 532, Alexa Fluor 549, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, tetramethylrhodamine, ATTO 532, ATTO Rho6G, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol l, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO590, ATTO Rhol3, or ATTO594.
EXAMPLES
[00158] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second( s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
Example 1
Materials and Methods (FIG. 2A-2B)
[00159] U2OS cells with a Cas9 HDR-generated HaloTag fusion to the endogenous NPM1 gene were cultured in Dulbecco’s modified Eagle medium (DMEM). Cells were stained for approximately 20 minutes at 37°C with either 50 pM JFX650 HaloTag ligand alone in DMEM or a mixture of 50 pM JFX650 HaloTag ligand and 10 nM JF549 HaloTag ligand in DMEM. After staining, cells were washed twice with lx phosphate buffered saline, and destained for 15 minutes in DMEM at 37°C before imaging. Cells were imaged with highly inclined and laminated optical sheet (HILO) illumination on an inverted Nikon TIRF microscope at a frame rate of 7.5 ms/frame. The imaging protocol consisted of 500-frame intervals of illumination with a 633 nm laser alternating with 60-frame intervals of illumination with a 561 nm laser. Particletracking software was used to count single molecule localizations, and the number of localizations at each frame index was averaged over multiple cells.
Results (FIG. 2A-2B)
[00160] Proximity-dependent photoactivation was investigated by imaging U2OS cells expressing an endogenous HaloTag fusion of the nucleolar protein NPM1, which forms pentameric complexes. Cells were incubated with a low concentration of JFX650 HaloTag ligand together with a 200-fold higher concentration of JF549 HaloTag ligand (FIG. 2A). Under these conditions, most NPMl-Halo pentamers that are labeled on one subunit with JFX650 are also labeled on other subunit(s) with JF549. Imaging with 633 nm light caused photobleaching or dark state conversion of the majority of JFX650 molecules, making it possible to visualize and count single molecules. Single-molecule imaging of JFX650 with 633 nm illumination was interspersed with 450 ms intervals of either 561 nm illumination (green points; depicted as grey points) or no illumination (blue points; depicted as black points). Reactivation of JFX650 molecules was observed following 561 nm illumination (grey points) but not following an equal time interval without illumination (black points). As a control, the same cell line was stained with JFX650 HaloTag ligand alone (FIG. 2B). No JFX650 reactivation was observed following 561 nm illumination (FIG. 2B, grey points), indicating that reactivation of JFX650 requires JF549.
Materials and Methods (FIG. 3A-3B)
[00161] U2OS cells were cultured in Dulbecco’s modified Eagle medium (DMEM). Half of the cells from a 10-cm culture dish were transiently transfected using the Lonza Nucleofector kit with 500 ng of an expression plasmid encoding either 1) a HaloTag fusion of a monomeric single-chain derivative of the bacterial Tet repressor (Halo-scTetR; Fig. 3 A; Kamionka et al., 2006, Nucleic Acids Research, DOI: 10.1093/nar/gkl316) or 2) a HaloTag fusion of dimeric Tet repressor (Halo-TetR; FIG. 3B). Halo-TetR protein dimers have two HaloTag moieties each, while Halo-scTetR protein monomers have only a single HaloTag moiety each. One day after transfection, cells were stained with a mixture of 50 nM Janelia Fluor 549 HaloTag ligand and 1 nM Janelia Fluor 646 HaloTag ligand in DMEM for 15 min at 37°C, washed twice with phosphate-buffered saline, and destained in DMEM for 15 min at 37°C. Cells were imaged with highly inclined and laminated optical sheet (HILO) illumination on an inverted Nikon TIRF microscope at a frame rate of 7.5 ms/frame. The imaging protocol consisted of 500-frame intervals of illumination with a 633 nm laser alternating with 5-frame intervals of illumination with either a 488 nm laser or 561 nm laser. Particle-tracking software was used to count single molecule localizations, and the number of localizations at each frame index was averaged over multiple cells.
Results (FIG. 3A-3B)
[00162] U2OS cells expressing either monomeric Halo-scTetR or dimeric Halo-TetR proteins were labeled with a small amount of JF646 HaloTag ligand and a 50-fold excess of JF549 HaloTag ligand. Under these conditions, it is expected that Halo-scTetR monomers were labeled with either JF549 or JF646, but not both, while most Halo-TetR dimers labeled with JF646 were also labeled with JF549. JF646 was imaged at a frame rate of 7.5 ms/frame using HILO illumination with a 633 nm laser. Single molecules could be resolved after the majority of JF646 molecules photobleached or transitioned into a reversible dark state. FIG. 3 shows the mean number of single-molecule JF646 localizations in each frame, averaged over multiple experimental replicates. Illumination at 633 nm was interspersed with brief 5-frame pulses of 488 nm light (black arrows; FIG. 3) or 561 nm light (grey arrows; FIG. 3). Illumination with 561 nm light (grey arrows and plot segments) reactivated JF646 for Halo-TetR dimers but not for Halo-scTetR monomers. This indicates that proximity between JF549 and JF646 within doublelabeled dimers is required for reactivation of JF646 by 561 nm light. By contrast, 488 nm illumination (black arrows and plot segments) reactivated JF646 for both monomers and dimers. Thus, reactivation by 488 nm light does not require proximity between JF549 and JF646 and can be used as an internal standard to which proximity-dependent reactivation by 561 nm light can be compared.
[00163] FIG. 6A-6C depict multiple cycles of alternating illumination. FIG. 6A depicts a scheme for quantification of the rate of proximity-dependent reactivation (e.g., by JF549 + 561 nm light), using direct reactivation of JF646 fluorophores with a shorter wavelength (e.g., 405 nm or 488 nm) as an internal standard. Open bar: 633 nm. The sample was imaged using 633 nm light and alternately stimulated with 405 or 488 nm light (to directly reactivate JF646) and 561 nm light (to induce proximity-dependent reactivation). FIG. 6B depicts a trace showing changes in the intensity of a sample in response to successive 561 nm (grey) and 488 nm (black) pulses. FIG. 6C: A rate constant can be determined by varying the duration of the green pulse (depicted in FIG. 6B as grey), while keeping the duration of the blue pulse (depicted in FIG. 6B as black) constant at 7 milliseconds (ms). The plot of reactivation ratio vs. green pulse duration is well fit by a single-exponential curve, consistent with first-order kinetics.
Materials and Methods (FIG. 4)
[00164] U2OS cells transfected with a TetR-Halo expression plasmid (as in Fig. 3) were fixed for 20 min at room temperature with 4% paraformaldehyde in lx phosphate buffered saline (lx PBS). Cells were rinsed twice with lx PBS, permeabilized for 45 min with 0.1% Triton X-100 in lx PBS, washed twice more with lx PBS, and stored overnight at 4°C. Fixed permeabilized cells were imaged at a frame rate of 7.5 ms/frame using HILO illumination, alternating between 500 frames of 633 nm illumination and 5 frames of 561 nm illumination. Single-molecule localizations were counted using particle tracking software, and the number of localizations at each frame index was averaged over multiple cells.
Results (FIG. 4)
[00165] Proximity-dependent photoactivation was assessed in fixed, permeabilized cells. U2OS cells expressing dimeric Halo-TetR were double-labeled with JF646 and JF549, fixed with paraformaldehyde, permeabilized with detergent, and incubated overnight in lx phosphate buffered saline. Reactivation of dark-state JF646 was observed upon illumination with 561 nm light (dashed vertical lines in FIG. 4), indicating that proximity-dependent photoactivation still occurs after cells are fixed and permeabilized. Materials and Methods (FIG. 5)
[00166] U2OS cells were electroporated with an expression plasmid encoding a fusion between the HaloTag and SNAPf tag proteins, connected by a flexible linker peptide with sequence GSGTGS. Cells were stained with 5 nM JFX650 SNAP ligand and 50 nM JF549 Halo ligand for 15 min at 37°C, washed twice with lx PBS, and destained for 15 min in DMEM. Cells were imaged with highly inclined and laminated optical sheet (HILO) illumination on an inverted Nikon TIRF microscope at a frame rate of 7.5 ms/frame. The imaging protocol consisted of 500- frame intervals of illumination with a 633 nm laser alternating with 1 -frame intervals of illumination with either a 488 nm laser (black in FIG. 5) or 561 nm laser (grey in FIG. 5). Particle-tracking software was used to count single molecule localizations, and the number of localizations at each frame index was averaged over multiple cells.
Results (FIG. 5)
[00167] A Halo-SNAP fusion protein was labeled with a mixture of JFX650 SNAP ligand and JF549 HaloTag ligand. SNAP-bound JFX650 was efficiently reactivated by 561 nm light in the presence of Halo-bound JF549. Thus, a combination of HaloTag and SNAP tag labeling can be used in the proximity-dependent photoactivation method.
[00168] Inclusion of a self-cleaving peptide between the Halo and the SNAP tags eliminates 561 nm photoactivation. The results are shown in FIG. 7. Reactivation in cis vs. reactivation in trans. As shown in the upper panel of FIG. 7, both direct (405 nm; black in FIG. 7) and indirect (561 nm; grey in FIG. 7) reactivation were observed when a combined Halo-SNAPf polypeptide was double-labeled with JF549 and JFX650. As shown in the lower panel of FIG. 7, only direct (405 nm) reactivation was observed when the two labels were separated by insertion of a self-cleaving peptide tag between Halo and SNAPf. This result provides another demonstration that reactivation of JFX650 by JF549 + 561 nm light depends on proximity of the two dyes within the same molecular complex.
[00169] FIG. 8 depicts application of proximity-dependent reactivation to detect inducible dimerization of JF549-labeled FRB-Halo and JFX650-labeled FKBP-SNAPf. U2OS cells were co-transfected with expression plasmids encoding the two proteins, labeled with JF549 Halo ligand and JFX650 SNAP ligand, and imaged using the alternating excitation protocol, either before or after addition of 333 nM rapamycin, which induces FRB-FKBP dimerization. An increase in the AGreen/ABlue (AGrey/ABlack) reactivation ratio was seen following rapamycin addition (shown are three experimental replicates). P-values were calculated using a 2-sided Student’s t-test. [00170] FIG. 9 depicts application of proximity-dependent reactivation to detect inducible dimerization of androgen receptor (AR) double-labeled with JF549 and JFX650. U2OS cells were co-transfected with expression plasmids encoding the Halo- AR and SNAPf-AR, labeled with JF549 Halo ligand and JFX650 SNAP ligand, and imaged using the alternating excitation protocol. Dihydrotestosterone (DHT) was added to a final concentration of 10 nM immediately after imaging cell #11. An increase in the AGreen/ABlue (AGrey/ABlack) reactivation ratio was seen over time as successive cells were imaged after DHT addition.
[00171] FIG. 10 depicts displacement histograms for 561 nm-reactivated, 405 nm-reactivated, and spontaneously reactivated molecules from the previous experiment (depicted in FIG. 9). Single molecules were tracked, and the resulting trajectories were analyzed using the Spot-On analysis package (Hansen et al. (2018) Elife 7:c33125) to infer the fraction of freely diffusing and bound molecules. The calculated bound fraction was substantially higher among 561 nm- reactivated molecules, consistent with a higher propensity of self-associated AR molecules to bind DNA than the overall mixture of monomeric and multimeric AR molecules.
[00172] FIG. 11A-11B show that proximity-dependent photoactivation can be used to detect differences in the bound fraction of distinct molecular subpopulations within the same cell. U2OS cells were transfected with a dual expression plasmid encoding a mixture of either A) Halo-SNAPf-tagged histone H2B (mostly bound) and SNAPf-3xNLS (mostly unbound; “3xNLS” denotes three repeats of the minimal SV40 nuclear localization sequence) or B) SNAPf-tagged histone H2B and SNAPf-Halo-3xNLS. Cells were labeled with JF549 Halo ligand and JFX650 SNAP ligand. Each cell was illuminated with 633 nm light to place JFX650 in a dark state and then imaged with 561 nm reactivation pulses followed by 405 nm reactivation pulses. Pairs of points show the bound fraction of 561 nm-reactivated and 405 nm-reactivated molecules for each cell. Molecules reactivated by 561 nm light have a higher bound fraction in (A) and a lower bound fraction in (B), consistent with selective reactivation of the doublelabeled component by 561 nm light.
[00173] FIG. 12 presents data indicating that proximity-dependent reactivation occurs over long distances. Insertion of a 67-amino acid rigid helical spacer between Halo and SNAPf led to little reduction in the reactivation of JFX650 by JF549. Because this spacer ostensibly contributes an additional 100-angstrom separation between Halo and SNAPf, this result suggests that the distance dependence of proximity-dependent photoactivation may be looser than that of singlemolecule FRET. Example 2
[00174] Single-molecule imaging has provided a new window into biochemical processes within live cells, yet a key limitation is the inability to visualize single-molecule trajectories while also detecting protein-protein interactions. A novel property of Janelia Fluor dyes is described herein: proximity-assisted photoactivation (PAPA), in which one fluorophore (the “sender” fluorophore) can reactivate a second fluorophore (the “receiver” fluorophore) from a dark state. PAPA requires proximity between the two fluorophores, yet its efficiency is less sensitive to inter-dye distance than Forster resonance energy transfer (FRET). It is shown herein that PAPA can be used in live cells both to detect protein-protein interactions and to enrich for a sub-population of labeled protein complexes in which two different dyes are in proximity. In proof-of-concept experiments, PAPA detected the expected correlation between androgen receptor self-association and chromatin binding within individual cells. These results establish a new way in which fluorophore photophysics can be harnessed to study molecular interactions in live cells by single-molecule imaging.
[00175] Here, it is shown that Janelia Fluor X 650 (JFX650) and similar fluorophores can be reactivated from a dark state by excitation of a nearby fluorophore such as Janelia Fluor 549 (JF549), a phenomenon that is termed herein “proximity-assisted photoactivation” (PAPA). In contrast to cyanine dye reactivation, PAPA of JF dyes occurs under physiological conditions in live cells and requires neither an oxygen scavenging system nor high concentrations of exogenous thiols. While PAPA requires proximity between the two fluorophores, its effective distance range extends beyond that of FRET, making it potentially a more versatile approach.
[00176] PAPA provides a way to detect protein complexes in live cells. It is shown herein that PAPA can be used to detect the formation of protein dimers and that it can selectively highlight sub-populations of molecules within defined mixtures. As a further proof of concept, singlemolecule tracking was combined with PAPA to analyze the increase in chromatin binding induced by self-association of androgen receptor. By enabling the previously elusive detection of protein-protein interactions, PAPA provides expanded information for live cell single-molecule imaging.
MATERIALS AND METHODS
Cell culture
[00177] U2OS cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose (ThermoFisher # 10566016), 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin at 37°C and 5% CO2. Phenol red-containing medium was used for propagation of cells, while phenol red-free medium (ThermoFisher # 21063029) was used to minimize fluorescence background in imaging experiments. Cloning
[00178] Ig linkers were derived from a previously reported plasmid containing repeats of the titin 191 Ig domain, which were codon-shuffled to prevent recombination (Scholl et al. (2016) Biomacromolecules 17:2502).
Stable transformation of cells and selection of clonal lines
[00179] To generate stable lines by PiggyBac integration, U2OS cells from a confluent 10-cm plate were trypsinized, resuspended in DMEM, and divided between two 15-ml conical tubes. Cells were centrifuged for 2 min at 200 g, and the medium was aspirated and replaced with 100 pl of Lonza Kit V transfection reagent (82 pl of Kit V solution and 18 pl of Supplement I) containing 1 pg of the donor plasmid and 1 pg of Super PiggyBac transposase plasmid. The cell suspension was transferred to an electroporation cuvette and electroporated using program X- 001 on the Amaxa Nucleofector II (Lonza). Cells in the cuvette were mixed with 300 pl of DMEM, and 100 pl of the suspension was plated in a 10-cm plate. After allowing cells to grow for 1-2 days, selection was initiated by adding puromycin to a final concentration of 1 pg/ml.
[00180] To generate clonal cell lines of Halo-mAR + SNAPf-mAR and FKBP-SNAPf-3xNLS + FRB-Halo-3xNLS, cells from a 10-cm plate were labeled with a mixture of 50 nM JF549 SNAP tag ligand and 50 nM JFX650 Halo tag ligand, and fluorescence-activated cell sorting (FACS) was used to sort single cells expressing both proteins into separate wells of a 96-well plate. For all other constructs, single-cell clones were obtained by limiting dilution into 96-well plates. Polyclonal pools of stably transfected cells were used for the other 2-component and 1- component plasmids in FIG. 20A-20F, FIG. 25A-25F, FIG. 22A-22H, and FIG. 23A-23I.
[00181] For the experiments in FIG. 17A-17B and FIG. 16A-16B, FACS was used to obtain polyclonal pools of U2OS cells expressing a low level of each Halo-linker-SNAPf construct. Confluent 10-cm plates of cells were stained with 50 nM each of JF549-STL and JFX650-HTL, and cells were sorted using the same intensity gate in the JFX650-Halo channel. Cells expressing pTG820 (Halo-3x Ig-SNAPf) and pTG828 (Halo-5x Ig-SNAPf) were sorted on a different day using the intensity of the previously sorted pTG747/U2OS pool to define a gate in the JFX650- Halo channel.
Visualization of fluorescently labeled proteins by SDS-PAGE
[00182] Cells in either 10-cm plates or 6-well plates were labeled with 500 nM of the indicated HTL or STL ligand for 1 h at 37°C, washed twice with lx phosphate buffered saline (PBS), trypsinized, and resuspended in DMEM. Cells were counted using a Countess 3 FL cell counter (Invitrogen), pelleted by centrifugation for 2 min at 200 g, and frozen at -80°C. Lysates were prepared by addition of 1 ml (for 10-cm plates) or 200 pl (for 6-well plates) of sodium dodecyl sulfate (SDS) lysis buffer without loading dye (Cattoglio et al. (2019), doi:10.7554/eLife.40164). Each lysate was passed through a 26-gauge needle 10 times to reduce its viscosity.
[00183] For the gels in FIG. 16A-16D and FIG. 16C-16B, samples of cell lysate corresponding to 100,000 cells were separated on a 10% SDS-PAGE gel, which was imaged on a Pharos FX imager (BioRad) using the “low-intensity” setting in the Cy3 channel. Cell lysate corresponding to 60,000 cells was loaded per lane of the gels in FIG. 19 A, FIG. 21A-21B, and FIG. 24A. The gel in FIG. 21A was imaged on a Pharos FX imager (BioRad) using the “low-intensity” setting in the Cy5 channel. The gels in FIG. 19A, FIG. 21B, and FIG. 24A were imaged in the 700 nm channel on an Odyssey imager (EI-COR) at 169 pm resolution with the “medium” quality setting and a z-offset of 0.5 mm. Precision Plus Protein All Blue Prestained Protein Standards (BioRad #1610373) were used as molecular weight standards for all gels.
Five-cell single-molecule imaging
[00184] One day prior to imaging, 25-mm No. 1.5H glass coverslips (Marienfeld, #0117650) were immersed in isopropanol, transferred with forceps to 6-well plates, and aspirated thoroughly to remove all traces of isopropanol. Cells were trypsinized, counted using a Countess 3 FL cell counter (Invitrogen), centrifuged for 2 min at 200 g, resuspended in phenol red-free DMEM, and plated at a density of 5 x 105 cells per well. Just prior to imaging, cells were incubated with Janelia Fluor HaloTag and SNAP tag ligands in phenol red-free DMEM for 15 min at 37°C, washed twice with lx phosphate buffered saline, and destained for at least 15 min in phenol red-free DMEM. The following dye concentrations were used for staining:
[00185] 1) 10 nM JF549-HTL and/or 250 pM JFX650-HTL for FIG. 13A-13B;
[00186] 2) 50 nM JF549-HTL and 5 nM JFX650-STL for FIG. 13C, FIG. 17A-17B, FIG. 18A-
18C, FIG. 20A-20F, FIG. 26A-26D, FIG. 25A-25F, FIG. 14B-14D, FIG. 19B, FIG. 22A-22H, FIG. 23A-23I, and FIG. 24B-24C;
[00187] 3) 50 pM JFX650 HTL or 5 nM JFX650-STL for FIG. 14A.
[00188] Coverslips were mounted in a stainless steel Attofluor™ Cell Chamber (ThermoFisher #A7816) and covered with 1 ml of phenol red-free DMEM with 10% FBS and penicillin/streptomycin. Cells were imaged using near-TIRF illumination on the microscope described in detail in [Hansen AS et al., Elife, 2018, PMID 29300163]. Laser power densities used for imaging were approximately 52 W/cm2 for 405 nm, 100 W/cm2 for 561 nm, and 2.3 kW/cm2 for 633 nm. Fluorescence emission was filtered through a Semrock 676/37 bandpass filter.
[00189] Cells were imaged at a rate of 7.5 ms/frame. Different experiments employed variations of an illumination sequence with alternating pulses of 633 nm red (R), 561 nm green (G), and 405 nm violet (V) light synchronized to the camera. These abbreviations are used, and the duration of the light pulse in brackets are indicated, below. For instance, “250 R [2 ms]” denotes 250 frames with a 2 ms pulse of red 633 nm illumination per frame. Red illumination was restricted to one 2 ms stroboscopic pulse per frame in single-molecule tracking to reduce the motion blur of moving molecules. To minimize tracking errors, the green and violet pulse durations were chosen to yield low numbers of localizations per frame after both green and violet reactivation pulses.
[00190] FIG. 13A-13B, FIG. 19B, and FIG. 24B: 10 cycles of 250 R [2 ms], 1 V [7 ms], 500 R [2 ms], 1 G [7 ms], 250 R [2 ms]
[00191] FIG. 13C and FIG. 17A-17B: 5 cycles of 100 R [7 ms], 1 V [7 ms] + R [7 ms], 200 R [7 ms], 1 G [7 ms] + R [7 ms], 100 R [7 ms]
[00192] FIG. 18A-18C: Same as in FIG. 13A-13B, but only 4 cycles.
[00193] FIG. 20A-20F, FIG. 22A-22D, and FIG. 23A-23F: Cells were first illuminated 10 s with 633 nm light to either photobleach or shelve most JFX650 fluorophores and then imaged with 10 cycles of 250 R [2 ms], 1 V [0.5 ms] + R [2 ms], 500 R [2 ms], 1 G [2 ms] + R [2 ms], 250 R [2 ms].
[00194] FIG. 25A-25F, FIG. 22E-22H, and FIG. 23G-23I: Same as FIG. 20A-20F, but with green pulses shortened to 0.5 ms.
[00195] FIG. 26B and FIG. 24B: Same as FIG. 13A-13B, but only 5 cycles.
[00196] FIG. 26C: Same as FIG.20A-20F, but with green pulses extended to 7 ms.
[00197] FIG. 26D: Same as FIG.20A-20F.
[00198] FIG. 14A: 10 cycles of 100 R [7 ms], 1 V [7 ms] + R [7 ms], 100 R [7 ms]. Only the first two cycles are shown in the figure.
[00199] FIG. 24C: Same as FIG. 20A-20F with green pulse duration set to 7 ms before DHT addition and 2 ms after DHT addition. DHT addition increases the rate of PAPA, i.e., the number of fluorophores reactivated by a green pulse of a given duration (FIG. 26B). For SPT experiments, the green pulse duration was thus shortened after DHT addition to keep the number of localizations per frame roughly equivalent and to prevent PAPA trajectories from becoming too dense for accurate tracking.
Analysis of ensemble PAPA experiments
[00200] For ensemble PAPA experiments, custom MATLAB code was used to sum the total intensity of all pixels in the field of view at each frame. Frame-by-frame intensity across multiple movies was averaged to obtain “sawtooth” plots of intensity vs. frame number. The PAPA/DR ratio was calculated by dividing the mean increase in fluorescence intensity induced by green and violet pulses.
Quantification of reactivation kinetics
[00201] To measure direct reactivation of JFX650-SNAPf as a function of 405 nm illumination time (FIG. 14B), cells expressing Halo-SNAPf-3xNLS (pTG747) were stained with 5 nM JFX650 STL and imaged at 7.5 ms/frame using a 5-phase protocol: 1) 20 frames with 1-ms pulses of 633 nm (intensity measurement before bleaching/shelving), 2) 400 frames with 7-ms pulses of 633 nm (bleaching/shelving), 3) 20 frames with 1-ms pulses of 633 nm (intensity measurement after bleaching/shelving), 4) N frames with 7-ms pulses of 405 nm (reactivation), 5) 20 frames with 1-ms pulses of 633 nm (intensity measurement after reactivation). The total pixel intensity was summed for all 20 frames in phases (1), (3), and (5), and the fractional reactivation was calculated by subtracting the increase in signal between (3) and (5) by the initial drop in signal between (1) and (3). The number of violet frames in phase 4, N, was varied as indicated by the values on the horizontal axis of FIG. 14B. The data were fitted to a singleexponential model. FLIM-FRET
[00202] Fluorescence lifetime was measured on a Zeiss LSM 980 microscope equipped with a Becker-Hickl SPC-150NX TCSPC module. Cells were labeled with a mixture of 50 nM JFX650- HTL and 50 nM JF549-STL for 1 h at 37°C, or with 50 nM JF549-STL alone as a no-FRET control. After briefly washing twice with lx PBS, cells were destained for at least 15 min prior to imaging. JF549 was excited using a 562 nm laser, and fluorescence emission was filtered through a Semrock 593/40 bandpass filter. Signal was acquired for 10 s over a 256 x 256 px region centered on each cell nucleus. Raw data were imported into SPCImage (Becker & Hickl) and fitted to a single exponential model without binning pixels. Decay constants and total fluorescence intensities for each pixel were exported in csv format. Custom MATLAB code was used to define nuclear masks by intensity thresholding and determine the mean fluorescence lifetime within the nucleus. FRET efficiency was calculated using the formula EFRET = 1-r/rO, where r is the fluorescence lifetime of the sample and r0 is the fluorescence lifetime of cells stained with JF549-STL alone.
Measuring mutual “occlusion” of PAPA and DR
[00203] To measure whether PAPA precludes DR and vice versa (FIG. 14C-14D), cells expressing Halo-SNAPf-3xNLS (pTG747) were labeled with 5 nM JFX650 STL and 50 nM JF549. The cells were then imaged at 7.5 ms/frame in 3 phases: 1) 500 frames of 633 nm light (7 ms pulses), alternating with unrecorded frames with either no illumination (black curves) or 7 ms pulses of 405 nm (grey curves) or 561 nm (dashed curves) illumination. 2) 20 frames with 7 ms pulses of 405 nm light (FIG. 14C) or 561 nm light (FIG. 14D). 3) 100 frames of 633 nm light (7 ms pulses).
Analysis of SPT data
[00204] Particles were localized and tracked using the quot package (https(://)github(dot)com/ alecheckert/quot). Custom MATLAB code (GitLab URL) was used to extract all trajectory segments occurring between 2 and 31 frames after pulses of 405 nm light (DR trajectories) and 561 nm light (PAPA trajectories). PAPA and DR trajectories were then separately analyzed using a Bayesian “fixed-state sampler” algorithm (https://github.com/alecheckert/spagl) (Heckert, et al. (2021) BioRxiv doi: https://doi.org/10.1101/2021.05.03.442482), which estimates the posterior probability distribution over a fixed array of diffusion coefficients. To allow a side- by-side comparison of the distributions for PAPA and DR trajectories, the same number of trajectories were included in the analysis for each. To this end, trajectories were randomly subsampled without replacement from whichever condition, PAPA or DR, had more trajectories. The fraction bound was calculated in FIG. 20C, FIG. 20F, FIG. 23C, and FIG. 23F by reanalyzing the data using a reduced 2-state model with diffusion coefficients 0.01 and 8.3 nf/s. The fraction slow-diffusing was calculated in FIG. 25C, FIG. 25F, and FIG. 231 by fitting to a 3-state model with diffusion coefficients 0.01, 2.1, and 13.2 p nr/s (FIG. 25C) or 0.01, 1.3, and 15.8 pm2/s (FIG. 25F and FIG. 231). Fraction bound for androgen receptor (FIG. 35C) was calculated by fitting to a 2-state model with diffusion coefficients 0.01 and 4.4 m2/s. Diffusion coefficients used in the reduced models correspond to the local maxima of the ensemble distributions. Displacement histograms in FIG. 22B, 22D, 22F, and 22H were tabulated using custom code in MATLAB.
RESULTS
Proximity-assisted photoactivation (PAPA) of Janelia Fluor dyes
[00205] PAPA was observed while imaging an oligomeric protein labeled with two different JF dyes. U2OS cells expressing Halo-tagged NPM1 (a pentameric nucleolar protein) (Heckert et al. (2021) BioRxiv, doi.org/10.1101/2021.05.03.442482) were labeled with a low concentration of Janelia Fluor X 650 Halo-tag ligand (JFX650-HTL)(20) to track single molecules, together with a higher concentration of Janelia Fluor 549 HaloTag ligand (JF549-HTL) to visualize nucleoli. When JFX650 was excited with red light (633 nm) and, alternately, JF549 was excited with green light (561 nm), it was noticed that some JFX650 molecules that had gone dark during red illumination suddenly reappeared after green illumination (green vertical lines in FIG. 13A (top panel) and FIG. 13B and green box in FIG. 13B). Reactivation of JFX650 by violet light was observed, both with and without JF549-HTL (violet vertical lines in FIG. 13A (top panel; middle panel) and violet box in FIG. 13B). However, reactivation of JFX650 by green light required co- labeling with JF549 (compare FIG. 13A (top panel; middle panel)), implying that reactivation results not from direct absorption of green light by dark-state JFX650 but indirectly due to excitation of JF549. Green illumination of cells labeled with JF549-HTL alone did not cause the appearance of localizations in the JFX650 channel, demonstrating that this effect is not due to JF549 photochromism FIG. 13A (bottom panel).
[00206] Because double-labeling of NPMl-Halo pentamers is expected to bring JF549 and JFX650 close together (FIG. 13A (top panel)), it was asked whether proximity of the dyes is required for reactivation. To test this, fusions of Halo and SNAPf separated by either a short flexible linker (Halo-SNAPf) or a tandem P2A-T2A “self-cleaving” peptide tag (Halo-PT2A- SNAPf) (Liu et al. (2017) Sci. Reports 7:2193) were expressed in U2OS cells (FIG. 13C), labeled cells with JF549-HTL and JFX650 SNAP tag ligand (JFX650-STL), and imaged JFX650 with red light interspersed with alternating pulses of violet and green light. While violet reactivation was similar for both constructs, green reactivation was substantially greater for Halo-SNAPf than for Halo-PT2A-SNAPf, implying that reactivation by green light requires proximity of the two dyes (FIG. 13C). This phenomenon is termed proximity-assisted photoactivation (PAPA). The dye that undergoes reactivation is called the “receiver” and the dye whose excitation induces reactivation is called the “sender”. The term “shelving” is adopted for conversion of the receiver into the dark state (Grimm et al. (2015) Nature Methods 12:244) and the term “direct reactivation” (DR) is adopted for reactivation by violet light.
[00207] The decline in JFX650 fluorescence was faster when the fluorophore was bound to SNAPf than when it was bound to Halo, and illumination with violet light reactivated a greater fraction of SNAPf-JFX650 than Halo-JFX650 (FIG. 14A). This suggests that shelving is more efficient for SNAPf-JFX650 than for Halo-JFX650, and so the receiver dye was coupled to SNAPf in subsequent experiments. Kinetic measurements indicate that about 10% of JFX650- SNAPf molecules enter the dark state under these experimental conditions (FIG. 14B). DR by violet light precluded subsequent PAPA by green light, and vice versa, implying that reactivation by both wavelengths occurs from a common dark state (FIG. 14C-14D). Other fluorophore pairs were tested, and it was found that PAPA occurred when tetramethylrhodamine (TMR), Janelia Fluor X 549 (JFX549), or Janelia Fluor 526 were used as the sender, or when JF646 or JFX646 were used as the receiver (FIG. 15A-15E).
[00208] FIG. 13A-13C. Proximity-assisted photoactivation (PAPA) of JFX650 by JF549. (A). Average number of localizations in the JFX650 channel as a function of frame number for heterozygous NPMl-Halo U2OS cells labeled with different combinations of JFX650-HTL and/or JF549-HTL. JFX650 molecules were excited with 633 nm light, interspersed with 7 ms pulses of 405 nm and 561 nm light (violet and green vertical lines). Right column: Schematic of NPM/NPMl-Halo pentamers labeled with JF549 (orange) and/or JFX650 (red). Reactivation of JFX650 by green light required labeling with JF549 (compare black arrows in (i) and (ii)). (B). Sample images of a single cell in the JFX650 channel. Leftmost panel: First movie frame prior to fluorophore bleaching/shelving. Green and violet boxes: Maximum intensity projection of all frames immediately before and after 561 nm (green) and 405 nm (violet) stimulation pulses, showing reactivation of molecules from the dark state. (C). Average fluorescence intensity in the JFX650 channel as a function of frame number in cells expressing a Halo-SNAPf fusion with a flexible linker (top panel; N = 40 cells) or a tandem P2A-T2A self-cleaving peptide separating Halo and SNAPf (PT2A; bottom panel; N = 20 cells). Halo was labeled with JF549-HTL and SNAPf with JFX650-STL. Reactivation by 405 nm light (violet lines) occurred in both cases, but reactivation by 561 nm light (green lines) was mostly eliminated by the self-cleaving peptide (compare black arrows).
[00209] FIG. 14A-14D. Properties of JFX650 reactivation. A) Comparison of darkening and reactivation of JFX650 bound to Halo (cyan) and SNAPf (black). Relative fluorescence intensity is plotted on the y-axis, averaged over multiple cells (n = 10 for Halo, 14 for SNAPf), and frame number is plotted on the x-axis. The frame rate was 7 ms/frame. Fluorescence intensity declined more rapidly for SNAPf-JFX650 than for Halo-JFX650. Violet pulses of 7 ms at frames 101 and 302 induced direct reactivation of JFX650, which was greater for JFX650-SNAPf than for JFX650-Halo. (B). Reactivation of SNAPf-JFX650 as a function of 405 nm pulse duration. SNAPf-JFX650 intensity was measured three times using 20 frames of 1 ms stroboscopic 633 nm illumination: 1) before darkening, 2) after darkening with 400 frames of non-stroboscopic (7 ms/frame) illumination, and 3) after reactivation by exposure to 405 nm pulses of varying duration. Percent reactivation was calculated by dividing the increase in intensity after reactivation by the decrease in intensity after darkening. Solid black curve shows a fit to a single-exponential model (black curve). (C) and (D). Mutual occlusion of green and violet reactivation. C) Halo-SNAPf-expressing U2OS cells labeled with JFX650-STL and JF549-HTL were imaged with 633 nm illumination alternating with unrecorded frames with either no illumination (black curve) or 405/561 nm illumination (grey/dashed curves). Integrated fluorescence intensity of JFX650 is plotted on the y-axis and frame number on the x-axis. JFX650 intensity initially declined for all conditions due to bleaching and darkening. A 20-frame (140-ms) pulse of 561 nm light was applied after frame 500 (grey rectangle). Although this reactivated JFX650 that had been exposed to red light only, it failed to reactivate JFX650 that had been exposed to alternating red and green or red and violet light. D) Same as C, but with a 20-frame (140-ms) violet pulse after frame 500. Some violet reactivation is still observed for cells exposed to alternating red and green light (dashed curve). This could indicate the presence of additional dark state(s) that can be reactivated by 405 nm light but not PAPA, although it might also reflect incomplete labeling of Halo by JF549 or photobleaching of JF549.
[00210] FIG. 15A-15E. PAPA between other sender-receiver pairs. U2OS cells expressing Halo-SNAPf-3xNLS were labeled with different SNAP tag ligand (STL) and HaloTag ligand (HTL) fluorophore combinations and imaged with red (633 nm) light alternating with 7-ms pulses of green (561 nm) and violet (405 nm) light. Fluorescence intensity averaged over multiple cells is plotted on the vertical axis and frame number is plotted on the horizontal axis. A) Tetramethylrhodamine (TMR)-HTL and JFX650-STL. B) Janelia Fluor X549 (JFX549)-HTL and Janelia Fluor 646 (JF646)-STL. C) Janelia Fluor 526 (JF526)-HTL and JFX650-STL. D) JFX650-STL-only control. E) JF549-only control.
Distance dependence of PAPA
[00211] To investigate how PAPA depends on sender-receiver distance, cell lines expressing fusion proteins in which Halo and SNAPf were separated by 0, 1, 3, 5, or 7 repeats of the titin 191 Ig domain were generated (22). U2OS cells were stably transfected with each transgene, and fluorescence-activated cell sorting (FACS) was used to select cells with similar low expression levels of each protein (FIG. 16A-16B; see Note 1).
[00212] Cells were labeled with a mixture of JF549-HTL and JFX650-STL and imaged as described above with red light interspersed with alternating pulses of violet light to induce DR and green light to induce PAPA. The ratio of the increase in fluorescence intensity in response to green and violet pulses (the “PAPA/DR ratio”) provides a normalized measure of PAPA efficiency, which corrects for cell-to-cell variability in the labeled protein concentration. For sufficiently short reactivation pulses, the PAPA/DR ratio increased linearly with the green pulse duration (with the violet pulse duration held constant), making it possible to measure relative rate constants by linear fitting (FIG. 17A-17B, left panel). In parallel, fluorescence lifetime imaging (FLIM) was used to measure FRET between JF549 and JFX650 for the same constructs (FIG. 17B, right panel; see Note 2).
[00213] Note 2. In principle, accurate quantification of FRET and PAPA does not require that the FRET donor or PAPA receiver be completely labeled. However, it is critical to achieve thorough labeling with the FRET acceptor and PAPA sender, as under-labeling would produce molecules labeled with donor or receiver only (i.e., with no FRET or PAPA), leading to an underestimate of the FRET or PAPA efficiency. Because Halo labeling is much more efficient than SNAPf labeling, Halo was labeled with the acceptor (JFX650-HTL) in FRET experiments and the sender (JF549-HTL) in PAPA experiments, while labeling SNAPf with the opposite fluorophore. [00214] As expected, FRET efficiency between JF549 and JFX650 declined sharply with increasing spacer length, from 0.271 ± 0.010 (95% CI) for the short linker to 0.124 ± 0.006 for a single Ig repeat and 0.020 ± 0.010 for three Ig repeats (FIG. 17B, right panel). FRET was essentially undetectable for 5 or 7 Ig repeats and for the PT2A self-cleaving peptide linker (FIG. 17B, right panel). In contrast, PAPA was observed for the 3x, 5x, and 7x Ig linker constructs (FIG. 17A-17B). The rate of photoactivation by green light declined gradually with increasing linker length yet was distinguishable from the background rate of the PT2A self-cleaving linker. These results indicate that PAPA has a more lenient dependence on inter-fluorophore distance than FRET.
[00215] FIG. 16A-16B. SDS-PAGE analysis of linker constructs. A-B) SDS-PAGE analysis of linker constructs expressed in U2OS cells and labeled with JF549-HTE (A) or JF549-STE (B). The amount of cell lysate loaded in each lane corresponds to 100,000 cells. Eookup table is set between 0 and 3000 counts for (A) and the top image in (B). The bottom image in (B) is the same gel with lookup table set between 0 and 800 counts to highlight faint bands. MW, molecular weight markers in kilodaltons. *, nonspecific bands. 0, unbound dye. A ladder of smaller fragments is present below the full-length protein for the larger linkers; these are predominantly labeled by Halo ligand but not SNAP ligand (see Note 1).
[00216] Note 1. Comparison of the SDS-PAGE gels in FIG. 16A and FIG. 16B shows that the Halo component of the self-cleaving Halo-PT2A-SNAPf fusion is present at a higher concentration than the SNAPf component. This is expected, given that ribosomes often terminate translation at self-cleaving peptide sequences without restarting translation of the next open reading frame (Hansen et al. (2018) eLife 7: doi:10.7554/eEife.33125). Although it was attempted to flow-sort populations of cells expressing similar levels of each protein, the Halo component of Halo-PT2A-SNAPf was expressed at a somewhat higher level than the other constructs (FIG. 16A). As a result, the reactivation rate observed for Halo-PT2A-SNAPf in FIG. 17A-17B likely overestimates the contribution of nonspecific background in PAPA measurements of the other constructs.
[00217] SDS-PAGE analysis of Halo-Ig-SNAPf linker constructs stained with Halo ligand revealed both a full-length protein and a regular ladder of faster-migrating bands, whose molecular weights correspond to Halo fused to different numbers of Ig repeats (FIG. 16A). SNAP ligand, in contrast, predominantly labeled the full-length protein (FIG. 16B); smaller fragments were only faintly visible with enhanced image contrast (FIG. 16B, lower panel). Because the FRET donor (JF549) and PAPA receiver (JFX650) were conjugated to SNAPf, which is almost exclusive to full-length polypeptides, it was expected that the presence of smaller Halo-tagged fragments will not impact the measurements of FRET and PAPA efficiency, as these Halo-only fragments will be “invisible” in measurements of JF549-STL fluorescence lifetime and JFX650-STL reactivation.
[00218] FIG 17A-17B. Comparison of distance-dependence of PAPA and FRET. (A). PAPA/DR ratio vs. green pulse duration for Halo-SNAPf fusions with a short, flexible linker or linkers containing different numbers of tandem Ig domains. Curves are linear fits (y = ax). Error bars, ± 1.96 * SE. PT2A, tandem P2A-T2A self-cleaving peptide. (B). Eeft panel: Reactivation rate of PAPA (slope of fits in (a) divided by the slope of the short linker construct). Right panel: FRET efficiency measured using fluorescence lifetime imaging (FEIM). Detection of inducible protein-protein interactions using PAPA
[00219] Based on the above results, it was reasoned that PAPA could be used to detect interaction of two different proteins labeled with SNAPf-JFX650 and Halo-JF549. As a test case, the rapamycin-inducible interaction of the proteins FRB and FKBP was monitored. U2OS cells expressing Halo-FRB and SNAPf-FKBP were labeled with JF549-HTE and JFX650-STE and imaged with alternating green and violet photostimulation as described above (FIG. 18 A and FIG. 19A-19B). Addition of rapamycin caused a dramatic increase in the ratio of PAPA (green reactivation) to DR (violet reactivation), consistent with ligand-induced dimerization of Halo- FRB and SNAPf-FKBP bringing together JF549 and JFX650 (FIG. 18B-18C and FIG. 19B)
[00220] FIG. 18A-18C. Detection of inducible dimerization using PAPA. (A). Halo-FRB was labeled with the sender fluorophore (JF549) and SNAPf-FKBP with the receiver fluorophore (JFX650). After shelving JFX650 with red (633 nm) light, DR and PAPA were alternately induced with pulses of violet (405 nm) and green (561 nm) light, respectively. Midway through the experiment, cells were treated with rapamycin to induce FRB-FKBP dimerization or with dimethylsulfoxide (DMSO) solvent as a negative control. (B). Ratio of fluorescence increase due to PAPA (green reactivation) and DR (violet reactivation) as a function of time after rapamycin addition. Black, rapamycin. Grey, DMSO solvent-only control. Individual data points represent single cells; solid lines show a 2-min moving average. (C). Average PAPA/DR ratio before (-) and after (+) addition of rapamycin (Rapa) or DMSO. Total number of cells: 75 before and 74 after rapamycin, 30 before and 30 after DMSO. Error bars, ± 2*SEM. Statistical significance was calculated using a 2-tailed t-test.
[00221] FIG. 19A-19B. SDS-PAGE and PAPA traces of FRB-FKBP. a) Fluorescence image of an SDS-PAGE gel of lysates from a clonal stable U2OS cell line expressing FKBP-SNAPf and FRB-Halo. Amount of lysate loaded in each lane corresponds to approximately 60,000 cells labeled with either JFX650-STE or JFX650-HTE. 0, unbound dye. b) Total fluorescence of the JFX650 receiver fluorophore as a function of frame number when illuminated with red light alternating with pulses of violet light to induce DR and green light to induce PAPA (vertical lines with lightning bolts). The top panel shows the average of cells imaged prior to rapamycin addition (n = 9), and the bottom panel shows the average of cells imaged between 5 and 15 min after rapamycin addition (n = 15). Fluorescence decreased initially due to bleaching and shelving of JFX650 and recovered upon fluorophore reactivation by green and violet light pulses. PAPA (green reactivation) was low prior to rapamycin addition (top panel) and increased following rapamycin addition (bottom panel). DR (violet reactivation) was observed both before and after rapamycin addition. AU, arbitrary units.
PAPA optically enriches a subset of molecules in defined 2-component mixtures
[00222] It was asked whether PAPA can be used to spotlight a sub-population of JFX650-labeled molecules in which JFX650 is close to JF549. As a simplified test case, defined mixtures of two proteins — one labeled with JFX650 only, and a second labeled with both JFX650 and JF549 — were analyzed. It was investigated whether PAPA could optically enrich the double-labeled component to distinguish its properties in single-molecule imaging.
[00223] First, SNAPf-tagged histone H2B (SNAPf-H2B), which is predominantly chromatinbound, was co-expressed along with a nuclearly localized Halo-SNAPf fusion (Halo-SNAPf- 3xNLS), which is mostly unbound (FIG. 20A)(2, 25). Cells were incubated with a mixture of JFX650-STL and JF549-HTL to label SNAPf-H2B with JFX650 only and Halo-SNAPf-3xNLS with both JFX650 and JF549 (FIG. 20A; FIG. 21A). JFX650 fluorophores were thoroughly photobleached/shelved using a 10-s pulse of intense red light, after which JFX650 was imaged with red light interspersed with pulses of green and violet light. After localizing and tracking single-molecule trajectories, those occurring after a green pulse (PAPA trajectories) were separated from those occurring after a violet pulse (DR trajectories) and a recently developed Bayesian state array SPT (saSPT) algorithm (Heckert, et al. (2021) BioRxiv doi: https://doi.org/10.1101/2021.05.03.442482) was applied to infer the underlying distribution of diffusion coefficients for each set of trajectories (its “diffusion spectrum” for short). As predicted, diffusion spectra revealed two peaks, one corresponding to bound molecules (D = 0.01 pm2/s, the minimum value in the state array), and one corresponding to freely diffusing molecules (D = 8.3 pm2/s). PAPA trajectories were enriched for freely diffusing molecules compared to DR trajectories, as expected if PAPA selectively reactivates JF549/JFX650 doublelabeled Halo-SNAPf-3xNLS molecules (FIG. 20B). Next, PAPA and DR trajectories from individual cells were re-analyzed using a 2-state model with bound (D = 0.01 pm2/s) and free (D = 8.3 pm2/s) states. Consistent with the ensemble analysis, PAPA trajectories had a lower bound fraction than DR trajectories in every cell (FIG. 20C). The same trend is apparent from comparison of particle displacement histograms and raw trajectories (FIG. 22A-22B). [00224] To exclude the possibility that enrichment of unbound molecules arose from a systematic bias in either the imaging or the analysis, the experiment was repeated with the reciprocal mixture of Halo-SNAPf-H2B and SNAPf-3xNLS (FIG. 20D). As expected, the opposite trend was observed; PAPA trajectories were enriched in bound molecules compared to DR trajectories, both across an ensemble of cells and at the single-cell level (FIG. 20E-20F and FIG. 22C-22D). As a further control, cells expressing either Halo-SNAPf-3xNLS or Halo- SNAPf-H2B alone were analyzed; as expected, PAPA and DR trajectories displayed virtually identical diffusion spectra for single components (FIG. 23A-23F).
[00225] Next, a defined mixture of two diffusing components was analyzed. Cytosolic Halo- SNAPf was co-expressed with a SNAPf-tagged synthetic protein that forms large, slowly diffusing 60-mers (Shia et al. (2016) Nature 535:136) (FIG. 25A), and imaging and analysis were performed as described above. Diffusion spectra showed the expected mixture of slow- diffusing (SNAPf-60-mer) and fast-diffusing (Halo-SNAPf) subpopulations (FIG. 25B). Compared to DR trajectories (violet curve), PAPA trajectories (green curve) were strongly enriched in the fast-diffusing subpopulation, consistent with selective reactivation by green light of the double-labeled Halo-SNAPf protein (FIG. 25B). The same trend was observed in individual cells (FIG. 25C) and was apparent from comparison of displacement histograms and raw trajectories (FIG. 22E-22F). The enrichment of the fast-diffusing population was not absolute, as a slow-diffusing peak was still observed among PAPA trajectories (FIG. 25B, solid black curve). As before, swapping SNAPf and Halo-SNAPf labels yielded the opposite trend, both at the ensemble and single-cell level (FIG. 25D-25F and FIG. 22G-22H). In this case, PAPA yielded especially strong enrichment of the slow-diffusing 60-mer peak (FIG. 25E-25F), which may reflect the proximity of each receiver molecule to multiple sender molecules within a Halo-SNAPf-tagged 60-mer.
[00226] Taken together, these results demonstrate that proximity-assisted photoactivation can be used to enrich a subpopulation of molecules in which a receiver fluorophore (e.g., JFX650) is in proximity to a sender fluorophore (e.g., JF549), thereby revealing the distinct properties of this subpopulation at both the ensemble and single-cell level.
[00227] FIG. 20A-20F. “Unmixing” of defined 2-component mixtures using PAPA. Left column (a,d): Schematic of different defined mixtures of two labeled proteins, in which one protein is labeled with JFX650 only and the other is labeled with both JFX650 and JF549. Center column (b,e): Inferred diffusion spectra of PAPA (green-reactivated) and DR (violet-reactivated) trajectories pooled from 20 cells. Right column (c,f): Fraction bound of PAPA and DR trajectories from individual cells, obtained from fits to a 2-state model. Paired, two-tailed t-tests of the comparisons in (c) and (f) showed both differences to be statistically significant with p = 6 x 109 and 7 x 10 8, respectively.
[00228] FIG. 21A-21B. SDS-PAGE gels of defined 2-component mixtures and 1-component controls. Fluorescent SDS-PAGE gels of lysates from stable U2OS cell lines expressing defined 2-component mixtures (1-4) and 1-component controls (5-6). Cells were labeled with either JFX650-STL (S) or JFX650-HTL (H) prior to lysis, and each lane was loaded with a volume of lysate corresponding to approximately 60,000 cells. Associated figure panels are listed after each construct. 0, unbound dye.
[00229] FIG. 22A-22H. Additional analyses of PAPA-fastSPT experiment with 2-component controls. a,c,e,g) A random subset of PAPA (green) and DR (violet) trajectories from individual cells. Trajectory centroids are aligned to a grid for visualization, and trajectories are displayed in order of increasing average displacement per step. Scale bar, 1 pm. b,d,f,h) Histograms of single-frame displacements for single-particle trajectories from all cells. Solid line, PAPA trajectories. Dashed line, DR trajectories. The sharp drop in frequency near 1 pm is due to the maximum displacement cutoff used in the particle tracking algorithm.
[00230] FIG. 23A-23I. PAPA-SPT analysis of single-component controls. Individual proteins fused with Halo-SNAPf were labeled with a mixture of JF549-HTL and JFX650-STL, imaged with alternating green and violet photostimulation pulses, and analyzed as in FIG. 20A020F. a-c) Halo-SNAPf-3xNLS. d-f) Halo-SNAPf-H2B. g-i) Halo-SNAPf-60-mer. Note that all 60 subunits are fused to Halo-SNAPf, but only a single label is shown for clarity. b,e,h) Inferred diffusion spectra of PAPA (green-reactivated) and DR (violet-reactivated) trajectories pooled from 10 cells (b,e,k). c,f,i) Fraction bound (c,f) or slow-diffusing (i) among PAPA and DR trajectories from individual cells, obtained from fits to a 2-state (c,f) or 3-state (i) model. P-values calculated using a two-sided paired t-test were 0.54 (c), 0.0080 (f), and 0.04 (1).
[00231] FIG. 25A-25F. “Unmixing” of defined 2-component mixtures using PAPA. Left column (a,d): Schematic of different defined mixtures of two labeled proteins, in which one protein is labeled with JFX650 only and the other is labeled with both JFX650 and JF549. Each subunit of the 60-mer is fused to SNAPf or Halo-SNAPf, though only one label is displayed for clarity. Center column (b,e): Inferred diffusion spectra of PAPA (green-reactivated) and DR (violet- reactivated) trajectories pooled from 20 cells (b) or 10 cells (e). Right column (c, f): Fraction slow-diffusing (i,l) of PAPA and DR trajectories from individual cells, obtained from fits to a 3- state model. Paired, two-tailed t-tests of the comparisons in (c) and (f) showed both differences to be statistically significant with p = 1 x 105 and 4 x 10 11 , respectively. Distinguishing the properties of androgen receptor monomers and dimers in single cells
[00232] As a proof-of-concept biological application, it was tested whether PAPA could be used to detect ligand-induced self-association of mammalian androgen receptor (AR) and distinguish the properties of AR monomers and dimer s/oligomers. First, SNAPf and Halo fusions of mouse AR were stably co-expressed in U2OS cells (which express very little endogenous AR (24)), the two proteins were labeled with a mixture of JFX650-STL and JF549-HTL (FIG. 24A), and the PAPA/DR ratio was measured by quantifying changes in JFX650 fluorescence intensity in response to alternating green and violet stimulation as described above. As expected, treatment with the androgen dihydrotestosterone (DHT) led to an increase in the ratio of PAPA to DR over the course of several minutes (FIG. 26B and FIG. 24B), consistent with the two fluorophores being brought together by ligand-induced interaction between SNAPf-mAR and Halo-mAR.
[00233] Next, PAPA was combined with single-molecule imaging to assess how self-association influences diffusion and chromatin binding by AR. Consistent with previous biochemical and live-cell imaging experiments, addition of DHT caused an increase in the overall bound fraction of AR (FIG. 26C-26D). Strikingly, PAPA trajectories had a higher bound fraction than DR trajectories, both before and after addition of DHT (FIG. 26C-26D). This supports a model in which self-association of AR increases its affinity for specific DNA sequence motifs in chromatin. Moreover, at least under the conditions of this experiment, a subset of self-associated AR molecules binds chromatin with elevated affinity even prior to addition of exogenous androgen. Thus, PAPA can be applied to monitor regulation of a biologically important proteinprotein interaction in live cells and discern its effect on chromatin binding.
[00234] FIG. 24A-24C. PAPA analysis of androgen receptor, a) Fluorescent SDS-PAGE gel of lysates from a clonal stable U2OS cell line expressing SNAPf-mAR and Halo-mAR. Cells were stained with JFX650-STL or JFX650-HTL, and a volume of lysate corresponding to 60,000 cells was loaded per lane. MW, molecular weight in kilodaltons, b) Ensemble PAPA analysis (see FIG. 19B legend) of interaction between SNAPf-mAR and Halo-mAR. PAPA signal (green reactivation) increased after addition of DHT. c) Fraction bound as a function of time relative DHT addition (vertical dashed line) for PAPA trajectories (filled markers, solid line) and DR trajectories (open markers, dashed line), based on fits to a 2-state model. Each data point corresponds to PAPA or DR trajectories from a single cell. Solid lines show moving averages over 10-min intervals.
[00235] FIG. 26A-26D. Analysis of mammalian androgen receptor using PAPA-SPT. a) Schematic of DHT-induced dimerization of JF549-Halo-mAR and JFX650-SNAPf-mAR. b) PAPA/DR ratio as a function of time relative DHT addition, c-d) Diffusion spectra of PAPA and DR trajectories, c) Before addition of DHT; N = 55 cells, d) After addition of DHT to a final concentration of 10 nM; N = 81 cells. Fraction bound was quantified by integrating the portion of each curve below D = 0.15 nf/s (vertical dashed line).
[00236] FIG. 27. Using PAPA to spotlight protein-protein interactions. 1) Label a SNAPf-tagged Target protein with a receiver fluorophore (e.g., JFX650) and a Halo-tagged Partner protein with a sender fluorophore (e.g., JF549). 2) Shelve the receiver fluorophore in the dark state using intense 633 nm illumination. Image receiver molecules with 633 nm light while alternately illuminating with 3) pulses of 561 nm light to induce proximity-assisted photoactivation (PAPA) of receiver-labeled Target molecules in complex with sender-labeled Partner molecules, and 4) pulses of 405 nm light to induce direct reactivation (DR) of receiver fluorophores, independent of proximity to the sender.
[00237] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is:
1. A method of detecting proximity of a first molecule to a second molecule, the method comprising: a) contacting the first molecule with the second molecule to form a contacted sample, wherein: i) the first molecule comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore in a proximity-dependent manner; and ii) the second molecule comprises the effector fluorophore, and wherein said contacting is carried out under conditions that inactivate the rhodamine dye; b) exciting the effector fluorophore with a second wavelength of light, wherein the excited effector fluorophore reactivates the inactivated rhodamine dye; and c) detecting fluorescence from the reactivated rhodamine dye in the contacted sample, wherein fluorescence from the reactivated rhodamine dye indicates that the first molecule and the second molecule are within about 20 nm of one another in the contacted sample.
2. The method of claim 1, wherein the rhodamine dye is inactivated at 633 nm.
3. The method of claim 1 or claim 2, wherein the rhodamine dye is a silicon rhodamine dye, optionally wherein the rhodamine dye is Janelia Fluor 646, Janelia Fluor X 646 or Janelia Fluor X 650.
4. The method of claim 1 , wherein the effector fluorophore is a second rhodamine dye or a fluorescent polypeptide.
5. The method of claim 4, wherein the effector fluorophore is JF526, JF549, JFX549, Alexa Fluor 532, Alexa Fluor 549, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, tetramethylrhodamine, ATTO 532, ATTO Rho6G, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol l, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO590, ATTO Rhol3, or ATTO594.
6. The method of any one of claims 1-5, wherein the first molecule is a biomolecule.
52
7. The method of any one of claims 1-6, wherein the second molecule is a biomolecule.
8. The method of claim 6 or claim 7, wherein: a) the first biomolecule is selected from the group consisting of a nucleic acid, a lipid, a carbohydrate, a small molecule, and a polypeptide; and b) the second biomolecule is selected from the group consisting of a nucleic acid, a lipid, a carbohydrate, a small molecule, and a polypeptide.
9. The method of any one of claims 6-8, wherein the first biomolecule is a polypeptide and wherein the second biomolecule is a polypeptide.
10. The method of any one of claims 6-8, wherein: i) the first biomolecule is a nucleic acid and wherein the second biomolecule is a polypeptide; or ii) the first biomolecule is a polypeptide and the second biomolecule is a nucleic acid; or iii) the first biomolecule is a nucleic acid and the second biomolecule is a nucleic acid.
11. The method of any one of claims 6-8, wherein: i) the first biomolecule is a lipid and wherein the second biomolecule is a polypeptide; or ii) the first biomolecule is a polypeptide and the second biomolecule is a lipid.
12. The method of any one of claims 6-8, wherein: i) the first biomolecule is a carbohydrate and wherein the second biomolecule is a polypeptide; or ii) the first biomolecule is a polypeptide and the second biomolecule is a carbohydrate.
13. The method of any one of claims 1-10, wherein the first molecule is a fusion polypeptide that comprises a heterologous fusion polypeptide selected from a SNAP, a CLIP, a HALO polypeptide, a dihydrofolate reductase (DHFR) polypeptide, a tetracysteine tag, a sortase polypeptide, or a transglutaminase polypeptide, and wherein the rhodamine dye is linked to the first molecule via the SNAP, CLIP, HALO polypeptide, DHFR polypeptide, tetracysteine tag, sortase polypeptide, or transglutaminase polypeptide.
14. The method of any one of claims 1-10, wherein the second molecule is a fusion polypeptide that comprises a heterologous fusion polypeptide selected from a SNAP, a CLIP, or a HALO
53 polypeptide, and wherein the effector fluorophore is linked to the first molecule via the SNAP, CLIP, or HALO polypeptide.
15. The method of any one of claims 1-14, wherein the first molecule and the second molecule are in vitro outside of a cell.
16. The method of any one of claims 1-14, wherein the first molecule and the second molecule are present in a cell in vitro.
17. The method of any one of claims 1-14, wherein the first molecule and the second molecule are present in a cell in vivo.
18. The method of claim 16 or claim 17, wherein the cell is a eukaryotic cell.
19. The method of claim 18, wherein the cell is present in a tissue or an organ.
20. The method of any one of claims 1-19, wherein said detecting is carried out using a microscope.
21. The method of any one of claims 1-20, further comprising reactivating the inactivated rhodamine dye by exposing the contacted sample to light of a wavelength of less than 500 nm, thereby activating the rhodamine dye in a proximity-independent manner.
22. The method of claim 21, comprising reactivating the inactivated rhodamine dye by exposing the contacted sample to light of a wavelength of from about 405 nm to about 488 nm.
23. A system comprising: a) a first molecule that comprises a rhodamine dye that can be inactivated at a first wavelength of light and activated to fluoresce by excitation of an effector fluorophore; b) a second molecule that comprises the effector fluorophore; and c) a light source that is configured to produce the first wavelength of light that inactivates a rhodamine dye.
24. The system of claim 23, wherein the light source is configured to produce a wavelength of light that excites the effector fluorophore.
54
25. The system of claim 23 or claim 24, wherein: a) the rhodamine dye is a silicon rhodamine dye, optionally wherein the rhodamine dye is Janelia Fluor 646, Janelia Fluor X 646 or Janelia Fluor X 650; and b) the effector fluorophore is a second rhodamine dye or a fluorescent polypeptide, optionally wherein the effector fluorophore is JF526, JF549, JFX549, Alexa Fluor 532, Alexa Fluor 549, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, tetramethylrhodamine, ATTO 532, ATTO Rho6G, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rholl, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO590, ATTO Rhol3, or ATTO594.
55
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