WO2025050003A1

WO2025050003A1 - High-throughput condensate modulator screen

Info

Publication number: WO2025050003A1
Application number: PCT/US2024/044811
Authority: WO
Inventors: Tuomas Pertti Jonathan KNOWLES; Shilpi ARORA; William Emrys Arter; Andrew Ciaran SEEBER; Kadi Liis SAAR; Marius REBMANN
Original assignee: Transition Bio, Inc.
Priority date: 2023-09-01
Filing date: 2024-08-30
Publication date: 2025-03-06

Abstract

The present invention is an ultra-high throughput droplet microfluidics platform that is possible to search through small-molecule chemical space to discover molecules directly through their signatures on protein phase behavior changes. Furthermore, the molecules discovered through this approach are modulators of phase transition characteristics of condensates or of LLPS of target macromolecules. These modulators induce desired functional and/or phenotypical changes, e.g., in cells and living systems. This approach allows the discovery of both stabilizers and destabilizers of FUS phase separation through screening phase diagrams in vitro. This invention establishes a new path forward to drugging a range of pathways dependent on biomolecular condensates, which have remained out of reach for conventional approaches.

Description

HIGH-THROUGHPUT CONDENSATE MODULATOR SCREEN

BACKGROUND OF THE INVENTION

Liquid-liquid phase separation of macromolecules leads to the organization of molecular contents and enables the crucial functionality of living cells. Due to their critical role in health and disease, finding modulators of liquid-liquid phase separation (LLPS) is becoming a major focus of drug discovery. These systems are particularly challenging targets, as they are highly coupled to other cellular processes and, as such, verifying target engagement from cellular screens has proven to be challenging. By contrast, molecular screens have the potential to solve this issue, but to date, the complexity of phase transition or liquid-liquid phase separation has eluded the development of such screening platforms. There remains a need for methodologies capable of identifying modulators of LLPS and other liquid-liquid phase transition characteristics, as well as assays for identifying biomolecules that are amenable to targeting by way of such modulators.

SUMMARY OF THE INVENTION

Herein, by developing an ultra-high throughput droplet microfluidics platform, the inventors show that it is possible to search through small-molecule chemical space to discover molecules directly through their signatures on protein phase behavior changes. Furthermore, the inventors show that molecules discovered through this approach are modulators of phase transition characteristics of condensates or of LLPS of target macromolecules. These modulators induce desired functional and/or phenotypical changes, e.g., in cells and living systems. The inventors illustrate this approach using, e.g., the protein FUS. The inventors demonstrate that this approach allows the discovery of both stabilizers and destabilizers of FUS phase separation through screening phase diagrams in vitro. Taken together, these findings establish a new path forward to drugging a range of pathways dependent on biomolecular condensates, which have remained out of reach for conventional approaches.

Accordingly, the present disclosure provides a method for screening a plurality of compounds to identify modulators of phase transition characteristics of a condensate and applications thereof.

The present disclosure provides a method for screening a plurality of compounds to identify condensate modulators that inhibit or promote LLPS of the target macromolecule, and applications thereof.

In some embodiment, the modulation of one or more phase characteristics of the condensate by the compound results in desired biological activity associated with the compound. In some embodiments, the inhibition or promotion of LLPS by the compound results in desired biological activity associated with the compound.

In some aspects of the application, methods of identifying compounds that modulate one or more phase transition characteristics of the condensate are provided. In some aspects, provided herein are methods of screening for compounds that modulate one or more phase transition characteristics of the condensate. In some aspects, provided herein are methods of high throughput screening and/or identifying compounds that modulate one or more phase transition characteristics of the condensate. In some aspects of the application, methods of identifying compounds that inhibit or promote LLPS of the target macromolecule are provided. In some aspects, provided herein are methods of screening for compounds that inhibit or promote LLPS of the target macromolecule. In some aspects, provided herein are methods of high throughput screening and/or identifying compounds that inhibit or promote LLPS of the target macromolecule.

In the first aspect, the disclosure provides a method of identifying condensate modulators, the method comprising: (a) producing a stream of microdroplets, wherein each of said microdroplets comprises a compound and the target macromolecule, wherein the concentration of the compound varies among the plurality of microdroplets; (b) determining, within each of the microdroplets, one or more phase transition characteristics of the condensate; and (c) determining whether the compound modulates one or more phase transition characteristics of the condensate, wherein: the determining is performed by comparing the concentration of the compound at which the one or more phase transition characteristics change to a concentration of a reference compound at which the one or more phase transition characteristics change.

In another aspect, the disclosure provides a method of identifying condensate modulators in vitro and in a cell, the method comprising: (a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, and wherein the concentration of the compound varies among the plurality of microdroplets; (b) determining, within each of the microdroplets, one or more phase transition characteristics of the condensate; and (c) determining whether the compound modulates one or more phase transition characteristics of the condensate, wherein: the determining is performed by comparing the concentration of the compound at which the one or more phase transition characteristics change as compared to a concentration of a reference compound at which the one or more phase transition characteristics change; (a’) introducing, into a cell, the compound identified in (c) as one that modulates one or more phase transition characteristics of the condensate in vitro, wherein the cell comprises the target macromolecule; and (b’) assessing whether the compound modulates one or more phase transition characteristics of the condensate in the cell, wherein: the determining is performed by comparing the concentration of the compound at which the one or more phase transition characteristics change in the cell, as compared to a concentration of a reference compound at which the one or more phase transition characteristics change in the cell.

In another aspect, the disclosure provides a method of identifying selective condensate modulators, the method comprising contacting a condensate modulator with a composition comprising a target macromolecule and a non-target macromolecule, and measuring one or more phase transition characteristics of the condensate, the method further comprising: (a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, a non-target macromolecule and wherein the concentration of the compound varies among the plurality of microdroplets; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-tar et), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, (b) determining, within each of the microdroplets, whether the target macromolecule has undergone phase transition, and whether the non-target macromolecule has undergone phase transition, thereby identifying the concentration of the compound at which the one or more phase transition characteristics change and the concentration of the compound at which the non-target macromolecule undergoes phase transition; (c) determining whether the compound selectively modulates one or more phase transition characteristics of the condensate, wherein: the determining is by comparing the concentration of the compound at which the one or more phase transition characteristics change as compared to a concentration of at which the non-target macromolecule undergoes phase transition.

In another aspect, the disclosure provides a method of identifying selective condensate modulators, the method comprising contacting a condensate modulator with a composition comprising a target macromolecule and a non-target macromolecule, and measuring one or more phase transition characteristics of the condensate, the method comprising:(a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, a non-target macromolecule and wherein the concentration of the compound varies among the plurality of microdroplets; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-target) , wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule; (b) determining, within each of the microdroplets, whether the target macromolecule has undergone phase transition, and whether the non-target macromolecule has undergone phase transition, thereby identifying the concentration of the compound at which the one or more phase transition characteristics change and the concentration of the compound at which the non-target macromolecule undergoes phase transition; (c) determining whether the compound selectively modulates one or more phase transition characteristics of the condensate, wherein: the determining is by comparing the concentration of the compound at which the one or more phase transition characteristics change as compared to a concentration of at which the non-target macromolecule undergoes phase transition, (a’) introducing, into a cell, the compound identified in (c) as one that selectively modulates one or more phase transition characteristics of the condensate, in vitro, wherein the cell comprises the target macromolecule and the non-target macromolecule; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-target), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, and (b’) assessing whether the compound selectively modulates one or more phase transition characteristics of the condensate in the cell, wherein: the determining is by comparing the concentration of the compound at which the one or more phase transition characteristics change in the cell, as compared to a concentration of the compound at which the non-target macromolecule undergoes phase transition in the cell.

In another aspect, the disclosure provides a method of identifying selective condensate modulators, the method comprising contacting a condensate modulator with a composition comprising a target macromolecule and a non-target macromolecule, and measuring one or more phase transition characteristics of the condensate in a cell, the method comprising: (a’) introducing, into a cell, the compound identified as one that selectively modulates one or more phase transition characteristics of the condensate, in vitro, wherein the cell comprises the target macromolecule and the non-target macromolecule; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-tar et), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, and (b’) assessing whether the compound selectively modulates one or more phase transition characteristics of the condensate in the cell, wherein: the determining is by comparing the concentration of the compound at which the one or more phase transition characteristics change in the cell, as compared to a concentration of the compound at which the non- target macromolecule undergoes phase transition in the cell.

In another aspect, the disclosure provides a method of identifying a compound that modulates one or more phase characteristics of a condensate in a cell, the method comprising: (a’) introducing, into a cell, the compound identified as one that modulates one or more phase transition characteristics of the condensate in vitro, wherein the cell comprises the target macromolecule; and (b’) assessing whether the compound modulates one or more phase transition characteristics of the condensate in the cell, wherein: the determining is by comparing the concentration of the compound at which the one or more phase transition characteristics change in the cell, as compared to a concentration of a reference compound at which the one or more phase transition characteristics change in the cell.

In another aspect, the disclosure provides a method of determining whether a compound inhibits or promotes LLPS of a target macromolecule in a cell, the method comprising: (a) introducing, into a cell, a compound that has been identified as one that promotes or inhibits LLPS of the target macromolecule in vitro, wherein the cell comprises the target macromolecule; and (b) assessing whether the compound promotes or inhibits LLPS of the target macromolecule in the cell, wherein: the assessing is performed by comparing the concentration of the compound at which the target macromolecule undergoes LLPS in the cell, as compared to a concentration of a reference compound at which the target macromolecule undergoes LLPS in the cell, the compound promotes LLPS in the cell when the concentration of the compound at which the target macromolecule undergoes LLPS in the cell is less than the concentration of the reference compound at which the target macromolecule undergoes LLPS in the cell, and the compound inhibits LLPS in the cell when the concentration of the compound at which the target macromolecule undergoes LLPS in the cell is greater than the concentration of the reference compound at which the target macromolecule undergoes LLPS in the cell.

In another aspect, the disclosure provides a method of determining whether a compound selectively inhibits or promotes LLPS of a target macromolecule in a cell, the method comprising: (a) introducing, into a cell, a compound that has been identified as one that selectively promotes or inhibits LLPS of the target macromolecule in vitro, wherein the cell comprises target macromolecule and a non-target macromolecule; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-tar et), wherein fluorescence of each target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, (b) assessing whether the compound selectively promotes or inhibits LLPS of the target macromolecule in the cell, wherein: the assessing is performed by comparing the concentration of the compound at which the target macromolecules undergo LLPS in the cell, as compared to a concentration of the compound at which the non-target undergo LLPS in the cell, the compound promotes LLPS in the cell when the concentration of the compound at which the target macromolecules undergo LLPS in the cell is less than the concentration of the compound at which the non-target macromolecule undergoes LLPS in the cell, and the compound inhibits LLPS in the cell when the concentration of the compound at which the target macromolecules undergo LLPS in the cell is greater than the concentration of the compound at which the non-target macromolecule undergoes LLPS in the cell.

In some embodiments, methods of identifying compounds that inhibit or promote LLPS of the target macromolecule are provided. In some aspects, provided herein are methods of screening for compounds that inhibit or promote LLPS of the target macromolecule. In some aspects, provided herein are methods of high-throughput screening and/or identifying compounds that inhibit or promote LLPS of the target macromolecule.

In the another aspect, the disclosure provides a method for screening a plurality of compounds to identify condensate modulators that inhibit or promote liquid-liquid LLPS (LLPS) of target macromolecules, the method comprising: (a) producing a stream of microdroplets, wherein each of said microdroplets comprises a compound and the target macromolecule, wherein the concentration of the compound varies among the plurality of microdroplets; (b) determining, within each of the microdroplets, whether the target macromolecule has undergone LLPS, thereby identifying the critical concentration (or concentration ranges) of the compound at which the target macromolecule undergoes LLPS; and (c) determining whether the compound promotes or inhibits LLPS, wherein: the determining is performed by comparing the concentration of the compound at which the target macromolecule undergoes LLPS as compared to a concentration of a reference compound at which the target macromolecule undergoes LLPS, the compound promotes LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is less than the concentration of the reference compound at which the target macromolecule undergoes LLPS, and the compound inhibits LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is greater than the concentration of the reference compound at which the target macromolecule undergoes LLPS.

In another aspect, the disclosure provides a method of determining whether a compound inhibits or promotes LLPS of a target macromolecule, the method comprising: (a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, and wherein the concentration of the compound varies among the plurality of microdroplets; (b) determining, within each of the microdroplets, whether the target macromolecule has undergone LLPS, thereby identifying the concentration of the compound at which the target macromolecule undergoes LLPS; (c) determining whether the compound promotes or inhibits LLPS, wherein: the determining is by comparing the concentration of the compound at which the target macromolecule undergoes LLPS as compared to a concentration of a reference compound at which the target macromolecule undergoes LLPS, the compound promotes LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is less than the concentration of the reference compound at which the target macromolecule undergoes LLPS, and the compound inhibits LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is greater than the concentration of the reference compound at which the target macromolecule undergoes LLPS, (a’) introducing, into a cell, the compound identified in (c) as one that promotes or inhibits LLPS of the target macromolecule in vitro, wherein the cell comprises the target macromolecule; and (b’) assessing whether the compound promotes or inhibits LLPS of the target macromolecule in the cell, wherein: the determining is by comparing the concentration of the compound at which the target macromolecule undergoes LLPS in the cell, as compared to a concentration of a reference compound at which the target macromolecule undergoes LLPS in the cell, and wherein the compound promotes LLPS in the cell when the concentration of the compound at which the target macromolecule undergoes LLPS in the cell is less than the concentration of the reference compound at which the target macromolecule undergoes LLPS in the cell, and the compound inhibits LLPS in the cell when the concentration of the compound at which the target macromolecule undergoes LLPS in the cell is greater than the concentration of the reference compound at which the target macromolecule undergoes LLPS in the cell.

In another aspect, the disclosure provides a method of determining whether a compound selectively inhibits or promotes LLPS of a target macromolecules, the method comprising: (a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, a non-target macromolecule and wherein the concentration of the compound varies among the plurality of microdroplets; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-tar et) , wherein fluorescence of each non-target fluorescent dye (FDnon-tar et) facilitates detection of one non-target macromolecule, (b) determining, within each of the microdroplets, whether the target macromolecule has undergone LLPS, and whether the non-target macromolecule has undergone LLPS, thereby identifying the concentration of the compound at which the target macromolecules undergo LLPS and the concentration of the compound at which the nontarget macromolecule undergoes LLPS; (c) determining whether the compound selectively promotes or inhibits LLPS of the target macromolecules, wherein: the determining is by comparing the concentration of the compound at which the target macromolecules undergo LLPS as compared to a concentration of at which the non-target macromolecule undergoes LLPS, the compound promotes LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is less than the concentration of the compound at which the non-target macromolecule undergoes LLPS, and the compound inhibits LLPS when the concentration of the compound at which the target macromolecules undergo LLPS is greater than the concentration of the compound at which the non- target macromolecule undergoes LLPS, (a’) introducing, into a cell, the compound identified in (c) as one that selectively promotes or inhibits LLPS of the target macromolecules in vitro, wherein the cell comprises the target macromolecule and the non-target macromolecule; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non- covalently bound to a fluorescent dye (FDnon-tar et), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, and (b’) assessing whether the compound selectively promotes or inhibits LLPS of the target macromolecule in the cell, wherein: the determining is by comparing the concentration of the compound at which the target macromolecules undergo LLPS in the cell, as compared to a concentration of the compound at which the non-target macromolecule undergoes LLPS in the cell, the compound promotes LLPS in the cell when the concentration of the compound at which target macromolecules undergoes LLPS in the cell is less than the concentration of the compound at which the non-target macromolecule undergoes LLPS in the cell, and the compound inhibits LLPS in the cell when the concentration of the compound at which the target macromolecules undergo LLPS in the cell is greater than the concentration of the compound at which the non-target macromolecule undergoes LLPS in the cell.

In another aspect, the disclosure provides a method of determining whether a compound inhibits or promotes LLPS of a target macromolecule in a cell, the method comprising: (a) introducing, into a cell, a compound that has been identified as one that promotes or inhibits LLPS of the target macromolecule in vitro, wherein the cell comprises the target macromolecule; and (b) assessing whether the compound promotes or inhibits LLPS of the target macromolecule in the cell, wherein: the assessing is performed by comparing the concentration of the compound at which the target macromolecule undergoes LLPS in the cell, as compared to a concentration of a reference compound at which the target macromolecule undergoes LLPS in the cell, the compound promotes LLPS in the cell when the concentration of the compound at which the target macromolecule undergoes LLPS in the cell is less than the concentration of the reference compound at which the target macromolecule undergoes LLPS in the cell, and the compound inhibits LLPS in the cell when the concentration of the compound at which the target macromolecule undergoes LLPS in the cell is greater than the concentration of the reference compound at which the target macromolecule undergoes LLPS in the cell. In another aspect, the disclosure provides a method of determining whether a compound selectively inhibits or promotes LLPS of a target macromolecule in a cell, the method comprising: (a) introducing, into a cell, a compound that has been identified as one that selectively promotes or inhibits LLPS of the target macromolecule in vitro, wherein the cell comprises target macromolecule and a non-target macromolecule; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-tar et), wherein fluorescence of each target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, (b) assessing whether the compound selectively promotes or inhibits LLPS of the target macromolecule in the cell, wherein: the assessing is performed by comparing the concentration of the compound at which the target macromolecules undergo LLPS in the cell, as compared to a concentration of the compound at which the non-target undergo LLPS in the cell, the compound promotes LLPS in the cell when the concentration of the compound at which the target macromolecules undergo LLPS in the cell is less than the concentration of the compound at which the non-target macromolecule undergoes LLPS in the cell, and the compound inhibits LLPS in the cell when the concentration of the compound at which the target macromolecules undergo LLPS in the cell is greater than the concentration of the compound at which the non-target macromolecule undergoes LLPS in the cell.

In some embodiments, the microdroplet is mounted on a microfluidic chip. In some embodiments, the microdroplet comprises various concentrations of the compound, and various concentration of the target macromolecule. In some embodiments, the microdroplet comprises buffer, salt solution and a trigger of LLPS or phase separation. In some embodiments, the microdroplet comprises a trigger of phase transition of a condensate that is known to modulate one or more phase transition characteristics of a condensate in vitro. In some embodiments, the microdroplet comprises a dye, e.g. Alexa 546 or Alexa 647 dyes. In some embodiments, the temperature of the microdroplets is varied by controlling the temperature of a channel in which the microdroplets flow. In some embodiments, the concentration of the compound, and the target macromolecule in the microdroplet is controlled by the flowrate. In some embodiments, the stream of microdroplets is a continuous stream.

In some embodiments, the trigger of phase transition is a protein, nucleic acid, salt, or polyethylene glycol (PEG), optionally wherein the PEG has an average molecular weight of from 600 Da to 20 kDa, optionally wherein the PEG has an average molecular weight of about 10 kDa. In some embodiments, the trigger is PEG1 Ok or a salt, such as NaCI or KCI, among others. In some embodiments, the trigger is a biological mixture, optionally wherein the biological mixture is a cell lysate. In some embodiments, the reference compound is DMSO or absent. In some embodiments, wherein the reference compound is a trigger of phase transition, the microdroplet comprises a different trigger of phase transition.

In some embodiments, the trigger of LLPS or phase separation is a protein, nucleic acid, salt, or polyethylene glycol (PEG), optionally wherein the PEG has an average molecular weight of from 600 Da to 20 kDa, optionally wherein the PEG has an average molecular weight of about 10 kDa. In some embodiments, the trigger is PEG10k or a salt, such as NaCI or KCI, among others. In some embodiments, the reference compound is DMSO or absent. In some embodiments, wherein the reference compound is a trigger of LLPS or phase separation, the microdroplet comprises a different trigger of LLPS or phase separation.

In some embodiments, the microdroplet comprises a trigger of phase transition that is known to promote or inhibit phase transition of the condensate in vitro. In some embodiments, the microdroplet comprises a trigger of phase transition that is known to promote or inhibit phase transition of the condensate in a cell. In some embodiments, the microdroplet comprises a trigger of phase transition that is known to promote or inhibit phase transition of the condensate. In some embodiments, the reference compound is a trigger of phase transition that is known to promote or inhibit phase transition of the condensate. In some embodiments, the reference compound is PEG10k.

In some embodiments, the microdroplet comprises a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule in vitro. In some embodiments, the microdroplet comprises a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule in a cell. In some embodiments, the reference compound is a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule. In some embodiments, the reference compound is PEG10k.

In some embodiments, the target macromolecule is a protein or nucleic acid. In some embodiments, the target macromolecule is DNA or RNA. In some embodiments, the target macromolecule is mRNA, hnRNA, or non-coding RNA. In some embodiments, the target macromolecule is rRNA, tRNA, IncRNSA, or miRNA. In some embodiments, the target macromolecule is mRNA, hnRNA, non-coding RNA, rRNA, tRNA, IncRNSA, or miRNA.

In some embodiments, the phase transition comprises homotypic phase transition, and the method results in identifying a compound as one that inhibits or promotes phase transition of the condensate. In some embodiments, the phase transition comprises heterotypic phase transition of the target macromolecule and one or more macromolecules. In some embodiments, the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the nontarget macromolecule, but wherein the compound is identified as one that modulates heterotypic phase separation of the target macromolecule and the non-target macromolecule. In some embodiments, the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that modulates phase transition characteristics of a condensate system that comprises the target macromolecule.

In some embodiments, the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that modulates heterotypic LLPS of the target macromolecule and the non-target macromolecule. In some embodiments, the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that phase separation of a condensate system that comprises the target macromolecule.

In some embodiments, the microdroplet comprises the target macromolecule and one or more macromolecules. In some embodiments, the one or more macromolecules are protein or nucleic acid. In some embodiments, the one or more macromolecules are DNA or RNA. In some embodiments, the one or more macromolecules are mRNA, hnRNA, or non-coding RNA. In some embodiments, the target macromolecule is rRNA, tRNA, IncRNSA, or miRNA. In some embodiments, the non-target macromolecule is a protein or a nucleic acid. In some embodiments, the non-target macromolecule is an RNA transcript, a specific RNA, a DMPK transcript, an AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

In some embodiments, the LLPS comprises homotypic LLPS, and the method results in identifying a compound as one that inhibits or promotes LLPS of the target macromolecule. In some embodiments, the LLPS comprises heterotypic LLPS of the target macromolecule and one or more macromolecules. In some embodiments, the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that phase separation of a condensate system that comprises the target macromolecule. In some embodiments, the microdroplet comprises the target macromolecule and one or more macromolecules. In some embodiments, the one or more macromolecules are protein or nucleic acid. In some embodiments, the one or more macromolecules are DNA or RNA. In some embodiments, the one or more macromolecules are mRNA, hnRNA, or non-coding RNA. In some embodiments, the target macromolecule is rRNA, tRNA, IncRNSA, or miRNA. In some embodiments, the non-target macromolecule is a protein or a nucleic acid. In some embodiments, the non-target macromolecule is an RNA transcript, a specific RNA, a DMPK transcript, an AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

In some embodiments, the method comprises the step of producing the microdroplets on a microfluidic chip. In some embodiments, each microdroplet is produced by mixing a portion of a stock mixture comprising the target macromolecule, and a portion of a stock mixture comprising the compound.

In some embodiments, each microdroplet comprises a portion of a stock mixture comprising the target macromolecule, a portion of a stock mixture comprising the compound and a portion of a stock mixture comprising a non-target macromolecule. In some embodiments, each microdroplet is produced by mixing a portion of a stock mixture comprising the target macromolecule, a portion of a stock mixture comprising the compound, and a portion of a stock mixture comprising the trigger. In some embodiments, each microdroplet is produced by mixing a portion of a stock mixture comprising the target macromolecule, a portion of a stock mixture comprising the compound, a portion of a stock mixture comprising a non-target macromolecule and a portion of a stock mixture comprising the trigger. In some embodiments, the mixing is automated mixing. In some embodiments, each microdroplet is produced by mixing a portion of a stock mixture comprising the target macromolecule, a portion of a stock mixture comprising the compound, and a portion of a stock mixture comprising the trigger.

In some embodiments, the stock mixture comprising the compound further comprises a fluorescent dye (FD_COm_Pound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration. In some embodiments, the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDco pound) correlates with the concentration of the compound and facilitates detection of the compound concentration; and the stock mixture comprising the trigger further comprises a fluorescent dye (FDtdgger) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDtdgger) correlates with the concentration of the trigger and facilitates detection of the trigger concentration.

In some embodiments, each fluorescent dye is different from one another. In some embodiments, the fluorescent dye (FDcompound), fluorescent dye (FDtarget) and fluorescent dye (FDtdgger) are different from one another. In some embodiments, the fluorescent dye (FDcompound), fluorescent dye (FDtarget), fluorescent dye (FDnon-target) and fluorescent dye (FDtdgger) are different from one another. In some embodiments, the fluorescent dye (FDcompound), fluorescent dye (FDtarget), and fluorescent dye (FDnon-target) are different from one another. In some embodiments, the fluorescent dye (FDcompound), and fluorescent dye (FDtarget) are different from one another.

In some embodiments, each fluorescent dye exhibits distinct excitation and emission spectra from one another. In some embodiments, the fluorescent dye (FDcompound), fluorescent dye (FDtarget) and fluorescent dye (FDtdgger) exhibit distinct excitation and emission spectra from one another. In some embodiments, the fluorescent dye (FDcompound), fluorescent dye (FDtarget), fluorescent dye (FDnon-target) and fluorescent dye (FDtdgger) exhibits distinct excitation and emission spectra from one another. In some embodiments, the fluorescent dye (FDcompound), fluorescent dye (FDtarget), and fluorescent dye (FDnon-target) exhibits distinct excitation and emission spectra from one another. In some embodiments, the fluorescent dye (FDcompound), and fluorescent dye (FDtarget) exhibits distinct excitation and emission spectra from one another.

In some embodiments, each fluorescent dye is selected from 7-nitrobenz-2-oxa-1 ,3-diazol-4- yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUE™ dye), 5- (dimethylamino)naphthalene-l -sulfonyl (Dansyl), pyrene, 7-amino-3-{[(2,5-dioxopyrrolidin-1 -yl)oxy]-2- oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350TM), 6,8-difluoro-7- hydroxy-4-methylcoumarin (MARINA BLUE™ dye), N-(2-aminoethyl)-4-{5-[4-(dimethylamino)phenyl]- 1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYL™ dye), 2,3,5,6-Tetramethyl-1 H,7H-pyrazolo[1 ,2- a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4-(ethylamino)naphthalen-2- yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUE™ dye), tris(N,N- diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrol idin-1 -yl)oxy]carbonyl}piperidin- 1 -yl)-2- oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405™), N,N-diethylethanaminium [9-{6-[(2,5- dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H- benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430™), 1 -[({4-[(7-nitro-2,1 ,3-benzoxadiazol- 4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSY™ dye), fluorescein, 2-(6-amino-3-iminio-4,5- disulfonato-3H-xanthen-9-yl)-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488™), 2’,7’-Difluoro-3’,6’-dihydroxy-3H-spiro[isobenzofuran-1 ,9’-xanthen]-3-one (OREGON GREEN™ 488), 1 ,3,5,7,8-pentamethyl-4,4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPY™ 493/503), rhodamine green, 13-[2-carboxy-3,4,6-trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 -yl)oxy-6- oxohexyl]amino]-2-oxoethyl]sulfanylphenyl]-7,7,9,17,19,19-hexamethyl-2-oxa-6,20- diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa-1 (14),3,5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546™), 2-[5-[3,3-dimethyl-5-sulfo-1 -(3-sulfopropyl)indol-1 -ium-2-yl]penta-2,4- dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5- sulfonic acid (ALEXA FLUOR 647™), and rhodamine red; and/or wherein one or more of the fluorescent dye is a fluorescent protein selected from GFP, mNeonGreen, mRFP, mCherry, and mPlum.

In some embodiments, fluorescent dye (FD_COm_Pound), fluorescent dye (FDtarget), and fluorescent dye (FDnon-target) are selected from 7-nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7- hydroxy-3-carboxycoumarin (PACIFIC BLUE™ dye), 5-(dimethylamino)naphthalene-1 -sulfonyl (Dansyl), pyrene, 7-amino-3-{[(2,5-dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}-4-methyl-2-oxo-2H-chromene- 6-sulfonic acid (ALEXA FLUOR 350™), 6,8-difluoro-7-hydroxy-4-methylcoumarin (MARINA BLUE™ dye), N-(2-aminoethyl)-4-{5-[4-(dimethylamino)phenyl]-1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYL™ dye), 2,3,5,6-Tetramethyl-1 H,7H-pyrazolo[1 ,2-a]pyrazole-1 ,7-dione (Bimane), 4-{[4- (Diethylamino)phenyl][4-(ethylamino)naphthalen-2-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 - iminium (CASCADE BLUE™ dye), tris(N,N-diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrolidin-1 - yl)oxy]carbonyl}piperidin-1 -yl)-2-oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405™), N,N- diethylethanaminium [9-{6-[(2,5-dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4- (trifluoromethyl)-8,9-dihydro-2H-benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430™), 1 - [({4-[(7-nitro-2,1 ,3-benzoxadiazol-4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSY™ dye), fluorescein, 2-(6-amino-3-iminio-4,5-disulfonato-3H-xanthen-9-yl)-5-((2, 3,5,6- tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488™), 2’,7’-Difluoro-3’,6’-dihydroxy-3H- spiro[isobenzofuran-1 ,9’-xanthen]-3-one (OREGON GREEN™ 488), 1 ,3,5,7,8-pentamethyl-4,4- difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPY™ 493/503), rhodamine green, 13-[2-carboxy-3,4,6- trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 -yl)oxy-6-oxohexyl]amino]-2-oxoethyl]sulfanylphenyl]- 7,7,9,17,19,19-hexamethyl-2-oxa-6,20-diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa- 1 (14), 3, 5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546™), 2-[5-[3,3-dimethyl-5-sulfo-1 - (3-sulfopropyl)indol-1 -ium-2-yl]penta-2,4-dienylidene]-3-methyl-3-[5-oxo-5-(6- phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5-sulfonic acid (ALEXA FLUOR 647™), and rhodamine red; and/or wherein one or more of the fluorescent dye is a fluorescent protein selected from GFP, mNeonGreen, mRFP, mCherry, and mPlum. In some embodiments, the target macromolecule is visualized by fluorescent microscopy to determine if the target macromolecule has undergone phase transition. In some embodiments, a finding that the target macromolecule is present within the microdroplet in discrete puncta is taken as an indication that the one or more phase characteristics of the condensate have changed, and a finding that the target macromolecule is present in a homogenous distribution within the microdroplet, absent discrete puncta, is taken as an indication one or more phase characteristics of the condensate have not changed.

In some embodiments, the target macromolecule is visualized by fluorescent microscopy to determine if the target macromolecule has undergone LLPS. In some embodiments, a finding that the target macromolecule is present within the microdroplet in discrete puncta is taken as an indication that the target macromolecule has undergone LLPS, and a finding that the target macromolecule is present in a homogenous distribution within the microdroplet, absent discrete puncta, is taken as an indication that the target macromolecule has not undergone LLPS.

In some embodiments, steps (a) through (c) are performed on a plurality of compounds, thereby screening a compound library to identify a compound that modulates the one or more phase transition characteristics of the condensate in vitro. In some embodiments, steps (a’) through (b’) have been performed on a plurality of compounds, thereby screening a compound library to identify a compound that modulates the one or more phase transition characteristics of the condensate in the cell. In some embodiments, the screening is high-throughput screening.

In some embodiments, steps (a) through (c) are performed on a plurality of compounds, thereby screening a compound library to identify a compound that inhibits or promotes LLPS of the target macromolecule in vitro. In some embodiments, steps (a’) through (b’) have been performed on a plurality of compounds, thereby screening a compound library to identify a compound that inhibits or promotes LLPS of the target macromolecule in the cell. In some embodiments, the screening is high- throughput screening.

In some embodiments, the compound library comprises from 10 to 100,000 compounds, or more. In some embodiments, the concentration of the compound at which the one or more phase transition characteristics change is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet, wherein the positive reference microdroplet comprises the target macromolecule and a trigger of phase transition, and the negative reference microdroplet comprises the target macromolecule. In some embodiments, the concentration of the compound at which the target macromolecule undergo phase transition is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet, wherein the positive reference microdroplet comprises the target macromolecule and a trigger of phase transition, and the negative reference microdroplet comprises the target macromolecule.

In some embodiments, the compound library comprises from 10 to 100,000 compounds, or more. In some embodiments, the concentration of the compound at which the target macromolecule undergoes LLPS is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet, wherein the positive reference microdroplet comprises the target macromolecule and a trigger of LLPS or phase separation, and the negative reference microdroplet comprises the target macromolecule. In some embodiments, the concentration of the compound at which the target macromolecule undergo LLPS is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet, wherein the positive reference microdroplet comprises the target macromolecule and a trigger of LLPS or phase separation, and the negative reference microdroplet comprises the target macromolecule.

In some embodiments, the compound is added to the cell as a stock mixture comprising the compound. In some embodiments, the trigger is added to the cell as a portion of a stock mixture comprising the trigger. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell naturally expresses the target macromolecule and undergoes LLPS. In some embodiments, the cell is induced to express the target macromolecule.

In some embodiments, the trigger is one or more agents that induces oxidative stress in the cell, thereby promoting localization of the target macromolecule to cytoplasmic stress granules. In some embodiments, the trigger that induces oxidative stress is sodium arsenite. In some embodiments, the trigger induces a change in a chemical modification state of the target macromolecule, thereby causing the target macromolecule to localize to a condensate in the cell. In some embodiments, the chemical modification is a post-transcriptional modification, such as phosphorylation, methylation, or acetylation. In some embodiments, the trigger that induces a change in methylation state is adenosine dialdehyde. In some embodiments, the trigger induces formation of nucleolar caps in the cell, thereby promoting localization of the target macromolecule to a nucleolar cap. In some embodiments, the trigger that induces formation of nucleolar caps in the cell is actinomycin D.

In some embodiments, step (b’) is performed by way of fluorescent microscopy. In some embodiments, the compound is identified as one that binds to, and/or modulates the activity of the target macromolecule. In some embodiments, the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that modulates heterotypic phase transition of the target macromolecule and the non-target macromolecule. In some embodiments, the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non- target macromolecule, but wherein the compound is identified as one that phase separation of a condensate system that comprises the target macromolecule. In some embodiments, the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4 ALK, FUS CHOP, YAP, TAZ or MBNL1 . In some embodiments, the non-target macromolecule is an RNA transcript, a specific RNA, a DMPK transcript, an AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

In some embodiments, step (b’) is performed by way of fluorescent microscopy. In some embodiments, the compound is identified as one that binds to, and/or modulates the activity of the target macromolecule. In some embodiments, the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that modulates heterotypic LLPS of the target macromolecule and the non-target macromolecule. In some embodiments, the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4 ALK, FUS CHOP, YAP, TAZ or MBNL1 . In some embodiments, the non-target macromolecule is an RNA transcript, a specific RNA, a DMPK transcript, an AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

In some embodiments, the method is performed on a plurality of compounds, thereby screening a compound library to identify one or more compounds that inhibit or promote phase transition of the target macromolecule in vitro. In some embodiments, the method is performed on a plurality of compounds, thereby screening a compound library to identify one or more compounds that inhibit or promote phase transition of the target macromolecule in the cell. In some embodiments, the screening is high-throughput screening.

In some embodiments, the method is performed on a plurality of compounds, thereby screening a compound library to identify one or more compounds that inhibit or promote LLPS of the target macromolecule in vitro. In some embodiments, the method is performed on a plurality of compounds, thereby screening a compound library to identify one or more compounds that inhibit or promote LLPS of the target macromolecule in the cell. In some embodiments, the screening is high-throughput screening.

In another aspect, the protein-RNA conjugate assay comprises (a) producing a composition that comprises a compound, and a target macromolecules wherein the target macromolecules is a protein, and a non-target macromolecule wherein the non-target macromolecule is RNA, wherein the concentration of the compound varies among the plurality of microdroplets; (b) determining, within each of the microdroplets, whether protein target macromolecule and/or RNA target macromolecule has undergone phase transition, thereby identifying the concentration of the compound at which each of the target macromolecules undergo phase transition; and (c) determining whether the compound promotes or inhibits phase transition, wherein the determining is performed by comparing the concentration of the compound at which either the protein target macromolecule and/or RNA macromolecule undergo phase transition as compared to a concentration of a reference compound at which either the protein target macromolecule and/or RNA macromolecule undergo phase transition, wherein the compound promotes phase transition when the concentration of the compound at which either the protein target macromolecule and/or RNA macromolecule undergo phase transition is less than the concentration of the reference compound at which either the protein target macromolecule and/or RNA macromolecule undergo phase transition, and wherein the compound inhibits phase transition when the concentration of the compound at which either the protein target macromolecule and/or RNA macromolecule undergo phase transition is greater than the concentration of the reference compound at which either the protein target macromolecule and/or RNA macromolecule undergoes phase transition. In some embodiments, the protein interacts with the RNA. In some embodiments, the compound modulates the interaction of the protein with the RNA. In another aspect, the protein-RNA conjugate assay comprises (a) producing a composition that comprises a compound, and a target macromolecules wherein the target macromolecules is a protein, and a non-target macromolecule wherein the non-target macromolecule is RNA, wherein the concentration of the compound varies among the plurality of microdroplets; (b) determining, within each of the microdroplets, whether protein target macromolecule and/or RNA target macromolecule has undergone LLPS, thereby identifying the concentration of the compound at which each of the target macromolecules undergo LLPS; and (c) determining whether the compound promotes or inhibits LLPS, wherein the determining is performed by comparing the concentration of the compound at which either the protein target macromolecule and/or RNA macromolecule undergo LLPS as compared to a concentration of a reference compound at which either the protein target macromolecule and/or RNA macromolecule undergo LLPS, wherein the compound promotes LLPS when the concentration of the compound at which either the protein target macromolecule and/or RNA macromolecule undergo LLPS is less than the concentration of the reference compound at which either the protein target macromolecule and/or RNA macromolecule undergo LLPS, and wherein the compound inhibits LLPS when the concentration of the compound at which either the protein target macromolecule and/or RNA macromolecule undergo LLPS is greater than the concentration of the reference compound at which either the protein target macromolecule and/or RNA macromolecule undergoes LLPS. In some embodiments, the protein interacts with the RNA. In some embodiments, the compound modulates the interaction of the protein with the RNA.

In some embodiments, the plurality of compounds comprises at least 100, 250, 500, 1000, 2000, 3000, 5000, 10000, 100000, or more different compounds. In some embodiments, the plurality of compounds is allowed to be in contact with the composition in separate reactions (e.g., separate vessels or wells). In some embodiments, the plurality of compounds is allowed to be in contact with the composition in separate reactions simultaneously.

In some embodiments, the methods further comprise repeating the steps of the method for a plurality of compounds. For example, in some embodiments, the methods comprise repeating the steps of the method for at least 2, 3, 4, 5, 10, 15, 20, 25, 40, 50, 75, 100, 250, 500, 1 ,000, 10,000, 100,000 or more compounds. In some embodiments, the method further comprises repeating the steps of the method with a plurality of concentrations of the compound.

In some embodiments, the methods described herein comprise producing a stream of microdroplets consisting of the compound and the target macromolecule which comprises one or more phases or a microdroplet capable or undergoing LLPS or phase transition. One of the ordinary skill in the art will readily recognize that biological processes, including the phase separation of a macromolecule, or the state of a condensate and components thereof, are dynamic. The methods described herein thus encompass producing a stream of microdroplets of the compound and the target macromolecule at any point in the life cycle of the one or more phases. For examples, the methods encompass producing a stream of microdroplets of the compound and the target macromolecule are present in any quantity, including being absent, are undergoing a morphological change, such as a change in size of liquidity, or are changing in composition. In some embodiments, the LLPS or phase transition occurs simultaneously after producing the stream of microdroplets. In some embodiments, the microdroplet is subject to a trigger prior to determining the LLPS or phase transition of the target macromolecule. In some embodiments, the microdroplet comprises a target macromolecule that has partially undergone LLPS or phase transition , and additional LLPS or phase transition occurs simultaneously with contacting the microdroplet with the microfluidic chip. In some embodiments, the microdroplet comprises a target macromolecule that has undergone LLPS or phase transition , and additional LLPS or phase transition occurs after contacting the microdroplet with the microfluidic chip. In some embodiments, the microdroplet comprises a target macromolecule that has partially undergone LLPS or phase transition , and reverses LLPS or phase transition simultaneously with and after contacting the microdroplet with the microfluidic chip. In some embodiments, the microdroplet comprises a target macromolecule that has undergone LLPS or phase transition , and reverses LLPS or phase transition simultaneously with and after contacting the microdroplet with the microfluidic chip.

In some embodiments, the target macromolecule undergoes LLPS or phase transition prior to contact with the microfluidic chip. In some embodiments, the method further comprises subjecting the microdroplet to a trigger prior to contacting the microfluidic chip. In some embodiments, the method further comprises contacting the microfluidic chip with a microdroplet comprising the target macromolecule that has undergone LLPS or phase transition , or with a microdroplet that is capable or undergoing LLPS or phase transition . In some embodiments, the LLPS or phase transition occurs simultaneously after producing the stream of microdroplets.

In some embodiments, the microdroplet is subject to a trigger prior to determining the LLPS of the target macromolecule. In some embodiments, the microdroplet comprises the target macromolecule that has undergone LLPS or phase transition , and additional LLPS or phase transition occurs simultaneously with contacting the microdroplet with the microfluidic chip. In some embodiments, the microdroplet comprises the target macromolecule that has not undergone LLPS or phase transition , and additional LLPS or phase transition occurs after contacting the microdroplet with the microfluidic chip. In some embodiments, the microdroplet comprises the target macromolecule that has partially undergone LLPS or phase transition , and additional LLPS or phase transition occurs simultaneously with and after contacting the microdroplet with the microfluidic chip.

In some embodiments, the microdroplet is subject to a trigger prior to determining the LLPS or phase transition of the target macromolecule. In some embodiments, the microdroplet comprises the target macromolecule that has undergone LLPS or phase transition , and additional LLPS or phase transition occurs simultaneously with contacting the microdroplet with the compound. In some embodiments, the microdroplet comprises the target macromolecule that has not undergone LLPS or phase transition , and additional LLPS or phase transition occurs after contacting the microdroplet with the compound. In some embodiments, the microdroplet comprises the target macromolecule that has partially undergone LLPS or phase transition , and additional LLPS or phase transition occurs simultaneously with and after contacting the microdroplet with the compound. In some embodiments, the microdroplet comprises the target macromolecule that has partially undergone LLPS or phase transition , and reverses LLPS or phase transition simultaneously with and after contacting the microdroplet with the compound. In some embodiments, the microdroplet comprises the target macromolecule that has undergone LLPS or phase transition and reverses LLPS or phase transition simultaneously with and after contacting the microdroplet with the compound.

In some embodiments, the methods described herein comprise contacting the microdroplet comprising the target macromolecule that has undergone LLPS or phase transition, or a microdroplet capable or undergoing LLPS or phase transition with a trigger of LLPS or phase separation or phase transition. In some embodiments, the microdroplet contains 0.5-6% of the trigger of LLPS or phase separation. In some embodiments, the trigger of LLPS or phase separation or phase transition is PEG1 Ok or a salt, such as NaCI or KCI, among others.

As described herein, the methods include producing a stream of microdroplets of the compound and the target macromolecule wherein (a) the microdroplet comprises the target macromolecule that has partially undergone LLPS or phase transition; and/or (b) the LLPS or phase transition occurs simultaneously with and/or after producing the stream of microdroplet. In some embodiments, the methods include producing a stream of microdroplets of the compound and the target macromolecule wherein (a) the microdroplet comprises the target macromolecule that has partially undergone LLPS; and/or (b) is capable of undergoing further LLPS or phase transition, wherein the LLPS or phase transition occurs simultaneously with and/or after producing the stream of microdroplets. In some embodiments, the methods described herein include producing a stream of microdroplets of the compound and the target macromolecule comprising one or more phases or a cellular composition capable or undergoing LLPS or phase transition.

In some embodiments, the conditions to undergo LLPS or phase transition comprises the addition of a trigger or the exposure of the cellular composition to a physical stressor. In some embodiments, the method comprises subjecting the microdroplet to any one or more of the following: (a) an oxidative stressor, (b) a mitochondrial electron transport chain inhibitor, (c) a heat stressor, (d) an osmotic stressor, € a hyperosmotic stressor (f) glycolysis inhibitor and (g) a salt solution. In some embodiments, the trigger is sodium arsenate, sorbitol, rotenone, 6-deoxyglucose in the absence of glucose, Actinomycin D, or Adenosine dialdehyde (AdOx). In some embodiments, the physical stressor is a heat stressor exposing the cellular composition to a temperature of 40-45°C, such as 43°C. In some embodiments, the physical stressor is an aging condition, e.g., incubation, shaking, or heat.

In some embodiments, the reference is a microdroplet that was treated with a reference compound. In some embodiments, the negative reference microdroplet is a microdroplet that does not comprise the compound. In some embodiments, the negative reference microdroplet is a microdroplet that was not treated with a trigger or physical stressor. In some embodiments, the negative reference microdroplet is prepared in a manner such that a meaningful result can be assessed for the compound. In some embodiments, the positive reference microdroplet is prepared in a manner such that a meaningful result can be assessed for the compound. For example, in some embodiments, the negative reference microdroplet is a microdroplet, wherein the microdroplet is prepared in a similar manner as the microdroplet comprising the compound, except the reference microdroplet does not comprise the compound. For example, in some embodiments, the positive reference microdroplet is a microdroplet, wherein the microdroplet is prepared in a similar manner as the microdroplet comprising the compound, except the reference microdroplet does not comprise the compound but comprises a trigger of LLPS or phase transition.

In some embodiments, the positive reference microdroplet comprises a trigger of LLPS or phase transition, the target macromolecule, non-target macromolecules, a phase-separation trigger, and buffer. In some embodiments, the trigger of LLPS or phase separation is 5% w/v 1 ,6-hexanediol.

In some embodiments, the negative reference microdroplet comprises a negative control compound, the target macromolecule, non-target macromolecules, a phase-separation trigger, and buffer. In some embodiments, the negative control compound is 1% v/v DMSO.

In some embodiments, determining one or more phase transition characteristics is based on any one or more of the following: (i) number of phases comprising and/or not comprising the target macromolecule; (ii) size of the one or more phases; (iii) location of the one or more phases; (iv) distribution of one or more phases; (v) surface area of the one or more phases; (vi) composition of the one or more phases, (vii) liquidity of the one or more phases; (viii) solidification of the one or more phases; (ix) dissolution of the one or more phases; (x) location of the target macromolecule; (xi) partitioning of the target macromolecule into the condensate; and (xii) aggregation of the target macromolecule.

In some embodiments, determining the LLPS of the target macromolecule is based on any one or more of the following: (i) number of phases comprising and/or not comprising the target macromolecule; (ii) size of the one or more phases; (iii) location of the one or more phases; (iv) distribution of one or more phases; (v) surface area of the one or more phases; (vi) composition of the one or more phases, (vii) liquidity of the one or more phases; (viii) solidification of the one or more phases; (ix) dissolution of the one or more phases; (x) location of the target macromolecule; (xi) partitioning of the target macromolecule into the condensate; and (xii) aggregation of the target macromolecule.

In some embodiments, the measuring is performed within about 60 days of the producing of the stream of microdroplets, about 21 days, about 14 days, about 7 days, about 2 days, about 1 day, about 12 hours, about 1 hour, about 30 minutes, about 15 minutes, about 1 minute, or about 30 seconds. In some embodiments, the determining is performed after about 5 seconds of the producing of the stream of microdroplets, such as, after about 15 seconds, about 1 minute, about 15 minutes, about 30 minutes, about 1 hour, about 12 hours, about 1 day, about 2 days, about 7 days, about 14 days, about 21 days, or about 60 days.

In some embodiments, the method further comprises repeating the measuring step of the method. For example, in some embodiments, the methods comprise repeating the measuring step of the method at least 2, 3, 4, 5, 10, or more times. In some embodiment, the methods comprise repeating the measuring step of the method on at least 1 , 2, 3, 4, 5, 10, or more microfluidic chips.

In some embodiments, the measuring step of the method is repeated after an interval of time, such as about 30 seconds, about 1 minute, about 15 minutes, about 1 hour, about 12 hours, about 1 day, about 5 days, about 7 days, about 14 days, about 21 days, about 60 days, or more. In some embodiments, the method further comprises comparing the LLPS over time, such as comparing the number of condensates with one day between measurements.

In some embodiments, the method is repeated with various concentrations of the compound. In some embodiments, the results from different concentrations of the same compound are compared. In some embodiments, the method is repeated with various concentrations of the one or more macromolecules. In some embodiments, the results from different concentrations of the same macromolecule are compared.

In some embodiments, the microdroplets are visualized by microscopy, including for example, fluorescence microscopy, epifluorescence microscopy, total internal reflection fluorescence microscopy, confocal microscopy, or multiphoton excitation microscopy. In some embodiment, the visualization is performed continuously on the stream of microdroplets. In some embodiments, the microdroplets and condensates are visualized by multi-color epifluorescence microscope. In some embodiments the multi-color epifluorescence microscope excites the microdroplets and detects a response. In some embodiments, the multi-color epifluorescence microscope excites and detects a response on multiple different fluorescence channels. In some embodiments, the multiple fluorescence channels are 470nm, 555nm and 640 nm. In some embodiments, the excitation is light via a laser diode. In some embodiments, the laser diode emits a wavelength of 470nm, 555nm, or 640 nm. In some embodiments, the relative concentration of the compounds and each of the target macromolecules are determined based on the respective responses of each target macromolecules to the excitation. In some embodiments, the relative concentration of the compounds is determined from the fluorescent intensity of Alexa 546 or Alexa 647 dyes. In some embodiments, the relative concentration of each of the target macromolecules comprise a different fluorophore which emits a specific wavelength in response to excitation.

In some embodiments, the multi-color epifluorescence microscope obtains a light-scattering profile of the microdroplet. In some embodiments, the light-scattering profile of the microdroplet determines the particular phases of the target macromolecules.

In some embodiments, the relative concentrations of each of the target macromolecules of the microdroplets are varied based on the measured relative concentrations of the target macromolecule, and the phases of the macromolecule present in the microdroplets. In some embodiments, the relative concentrations of the constituents of the microdroplets are systematically varied.

In some embodiments, the target macromolecule may be labeled, for example, labeled with a fluorophore. In some embodiments, the target macromolecule comprises a fluorescent dye, such as a fluorescent protein, e.g., GFP, RYP, or YFP. In some embodiments, the method comprises contacting at least a portion of the microdroplet with a label. In some embodiments, the label is a labeled binding molecule, such as an antibody or biotin-binding protein. In some embodiments, the label is a stain, such as a stain specific to an organelle. In some embodiments, each of the target macromolecules is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-target) , wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule. In some embodiments, each of the target macromolecules is, independently, covalently, or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule.

In some embodiments, the method further comprises imaging at least a portion of the microdroplet, such as a field of view. In some embodiments, the method further comprises a contacting at least a portion of the microdroplet with a stain. In some embodiments, the method further comprises contacting at least a portion of the microdroplet with a DNA-damaging condition. In some embodiments, the DNA-damaging condition is laser irradiation.

In some embodiments, the method comprises constructing a phase diagram. In some embodiments, the phase diagram is constructed on a drop-by-drop basis. In some embodiments, the phase of the target macromolecule is determined based on characteristics of the image indicative of particular phases.

The LLPS or phase transition of target macromolecules can be determined for a portion or all of the microdroplet. Accordingly, in some embodiments, the method comprises determining the LLPS or phase transition of the target macromolecule into LLPS or phase transition in a portion of the microdroplet. In some embodiments, the method comprises determining the LLPS or phase transition of the target macromolecule in the entire microdroplet.

In some embodiments, the compound is a small molecule, a polypeptide, a lipid, or a nucleic acid. In some embodiments, the compound is an approved compound, such as a compound approved for medical treatment by the United States Food and Drug Administration. In some embodiments, the compound is a novel compound. In some embodiments, the compound is charged. In some embodiments, the compound is hydrophobic. In some embodiments, the compound is hydrophilic. In some embodiments, the compound is a small molecule. In some embodiments, the small molecule is an alkaloid, a glycoside, a phenazine, a phenol, a polyketide, a terpene, or a tetrapyrrole. In some embodiments, the compound is an antibody. In some embodiments, the compound is a nucleic acid. In some embodiments, the compound is RNA, such as a siRNA, miRNA, or mRNA. In some embodiments, the compound is a non-naturally occurring compound. In some embodiments, the compound is a naturally occurring compound. When a plurality of candidate compounds is used for screening, the plurality of candidate compounds can be of the same type or of different types.

In another aspect, the disclosure provides a method of identifying a compound useful for treating a disease in an individual in need thereof, wherein the LLPS or phase transition of the target macromolecule is associated with the disease, and wherein the compound is identified as one that inhibits the LLPS or phase transition of the target macromolecule, thereby identifying a compound useful for treating the disease. In some embodiments, the method includes the step of administering a therapeutically effective amount of the compound to an individual diagnosed as having the disease.

In another aspect, the disclosure provides a method for identifying target macromolecules, the method comprising: (a) in silico screening a multimodal data set from a plurality of human biological samples to identify a plurality of genetic variations that distinguish a human disease state from a human healthy state;(b) in silico screening a multimodal data set from a plurality of in vitro disease relevant cell line models to identify a plurality of genetic variations that distinguish a disease state from a healthy state; (c) analyzing the plurality of genetic variations to identify one or a subset of genetic variations that lead to a change in concentration of the target macromolecule, a chemical alternation of the target macromolecule, and/or a change in the endogenous environment of the target macromolecule; (a’) determining that the change in concentration, the chemical alteration and/or change in endogenous environment of the target macromolecule leads to aberrant condensation behavior in the disease-associated model; and (b’) determining that the change described in (a’) does not lead to an aberrant condensation behavior in biological samples from healthy volunteers or nondiseased cell models.

In some embodiments, the target macromolecule is a protein capable of undergoing LLPS or phase transition, or localizing into biomolecular condensates. In some embodiments, the multimodal data modalities comprise one or many of the following: DNA sequencing data describing genetic alterations, transcriptomic data, proteomic data, dependency data acquired by measuring cell viability upon knockdown or knockout of the target gene of interest. In some embodiments, the chemical alternation of the target macromolecule comprises one or more mutations in the protein. In some embodiments, the one or more mutations in the protein is missense mutations, deletions, fusions, truncations, and/or frameshift mutations. In some embodiments, the chemical alternation of the target macromolecule comprises post-translational modifications. In some embodiments, the post- translational modification is phosphorylation, acetylation, sumoylation, ubiquitination, myristoylation, and/or palmitoylation.

In some embodiments, one or more of the mutations are enriched in one or more regions of a protein that are associated with LLPS. In some embodiments, the one or more regions of a protein are a functional domain of a protein, a low complexity region, or a disordered region of the protein. In some embodiments, the one or more regions of a protein that are associated with LLPS is based on experimentally obtained wet lab data. In some embodiments, the one or more regions of a protein that are associated with LLPS is based on statistical analysis of the enrichment of protein domains into biomolecular condensate systems using the composition of previously characterized condensate systems as the input. In some embodiments, the one or more regions of a protein that are associated with LLPS is determined by using predictive models that link a protein sequence and its altered form to the condensation propensity. In some embodiments, the one or more regions of a protein that are associated with LLPS is determined via a saturation mutagenesis analysis across the sequence that identifies regions of the protein wherein condensation behavior is sensitive to alterations in the sequence.

In some embodiments, the change in concentration as a result of the genetic variations leads to an altered condensation state of the protein as can be deduced from a comparison to the endogenous saturation concentration (c_Sat) of the target macromolecule. In some embodiments, the genetic variations cause the concentration of the macromolecule to increase above the endogenous saturation concentration. In some embodiments, the genetic variations that lead to an increased concentration of the target macromolecule are a fusion of two genes, a missense or truncation mutation that results in reduced degradation of the target macromolecule.

In some embodiments, the genetic variations cause the concentration of the macromolecule to reduce below the endogenous saturation concentration. In some embodiments, the genetic variations that lead to a decrease in the target macromolecule are a deletion mutation, a missense or truncations mutation that result in reduced rate of protein production.

In some embodiments, the change in the endogenous environment of the target macromolecule comprises a change in the availability of one or more endogenous modulators of LLPS. In some embodiments, the change in the endogenous environment of the target macromolecule comprises a change in the level of molecular crowding. In some embodiments, the change in the endogenous environment comprises a reduction in the concentration of the key effectors of LLPS. In some embodiments, the key effectors of LLPS are scaffolding proteins or RNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the platform that can identify hits that can modulate condensate targets of interest using an in vitro (molecular) screen and result in the discovery of hits that are specific to the condensate system of interest (#1 ), cover a variety of molecular modalities (#2), show target engagement (condensate modulation) in cells (#3). The hits show functional effects in cells (#4) and animal models (#5) in agreement with the disease hypothesis. The condensate target of interest may be identified via in silico methods (#6) as outlined in more detail in upcoming figures.

FIG. 2 is a graph showing the effects on the phase boundary of condensate systems (EML4- ALK DDX3X, G3BP1 , YAP, or YTHDC1 ) by Compound A, as shown in Example 1 , below, and demonstrates that the compounds identified from the in vitro I molecular screen are specific (Fig. 1 , box #1 ). The data show that a hit identified against the EML4-ALK condensate is selective over other condensate systems (DDX3X, G3BP1 , YAP, YTHDC1 ).

FIGS. 3A-3C are a series of graphs illustrating that the Molecular condensate assay can identify hits with a variety of different binding modes (box #2). Three compounds (Compound B, Compound C, Compound D) tested for their modulation of G3BP1 -RNA condensates as measured by a microfluidics- based condensate assay (PhaseScan™). All the compounds modulate condensates (left column). Only one of the compounds (Compound C) bind the protein directly as measured by a microscale thermophoresis assay (MST; middle column). Only one of the compounds (Compound D) disrupts the protein-RNA interaction as identified by microfluidic diffusional sizing (MDS; right panel). The phase boundary shifts of G3BP1 -RNA condensates by three compounds (Compound B, Compound C, Compound D), the binding affinity of the compound to the protein (as measured by microscale thermophoresis assay), and the disruption of a protein-RNA interaction by the compound (as measured by microfluidic diffusional sizing).

FIG. 3D is a chart demonstrating that the PhaseScan™ method disclosed herein can identify targets that modulate phase transitions through either protein-binding or protein-RNA disruption and this data highlights that the molecular screening assay, here reduced to practice via the use of the microfluidics-based screening assay, can identify hits across a variety of binding mechanisms. FIGS. 4A-4B are a series of graphs illustrating the compounds identified from the in vitro screening assay modulate cellular condensates and of the phase transition of Suramin and DMSO as described in Example 3, below. A compound (suramin) identified from the in vitro screening assay leads to the dissolution of FUS condensates in an in vitro screening assay (left) relative to DMSO control (right).

FIG. 4C is a series of images of the actinomycin induced FUS condensates in HeLa cells that are treated with DMSO control, or 5 pM or 20 pM Suramin, as described in Example 4, below. Suramin prevents the formation of actinomycin induced FUS condensates, specifically nucleolar caps, in HeLa cells (cyan). Data shown for the cases when the cells are treated with 5 pM and 20 pM dose of suramin. These data highlight the capability of the in vitro screen to identify hits that lead to condensate dissolution in cells.

FIG. 5 is a series of graphs showing that identified hits lead to desired functional effects in cells through the effects on RNAseq of Suramin on actinomycin induced FUS condensates in HeLa cells, as described in Example 5, below. RNAseq data of Actinomycin D treated HeLa cells highlight the downregulation of genetic pathways associated with RNA processing, the nucleolus, gene silencing and negative regulation of gene expression, which is in agreement with its previously confirmed mechanism of action. Treatment with suramin, the compound identified from the molecular screening assay (Figure. 4), leads to upregulation of these genes that contributes towards the rescue of the desired phenotype.

FIG. 6 is a series of images of FUS condensates in P525L and R495X cells that are treated with DMSO control and 500 pM Suramin as described in Example 6, below. FUS mutations common in ALS (P525L and R495X) lead to the formation of condensates that are more resistant to suramin treatment, highlighting the key role that the condensates play in the disease.

FIG. 7 is a series of images showing that the molecular I in vitro screening process can be performed on a microdroplet-based platform that allows high-throughput quantification of the phase space and thereby effective determination of the location of phase boundaries and the experimental setup for microfluidic droplet generation and imaging. Fluids are injected into a flow-focusing microfluidic drop generator, including the sequential injection of compounds from a well-plate autosampler, and microdroplets are incubated on a chip before undergoing simultaneous three-color imaging (as is described in Example 7, below) and a series of phase diagrams and normalized phase boundary shifts relative to DMSO control of FUS in the presence of 1029 compounds from an FDA-approved repurposing library (as is described in Example 8, below).

Phase separation is probed inside microdroplets where each droplet contains reagents under different conditions (e.g. different protein, RNA, modulators, such as PEG, salt, temperature etc.) The conditions may be barcoded via the use of fluorescent dyes. By determining if each droplet represents a homogenous (blue) or a phase separated (red) state, a multidimensional phase diagram can be constructed. Thousands of compounds can be probed in the assay and the phase separation boundary is determined for each compound to identify modulators of interest (e.g. enhancers, preventers).

FIG. 8 is a schematic diagram of an exemplary silico screening process described herein. The biomolecular condensate target screened in the molecular / in vitro assay (FIG. 1 , box #6) may be identified via an in-silico screening process that brings omics data from human samples and cellular models together with biomolecular condensate related data, such as data acquired via imaging, spatial transcriptomics I proteomics, and biochemical assays, among others. The latter data can be used to develop models that predict the condensation behavior of targets which do not have their condensation behavior profiled, their mutated forms or under conditions (e.g. cell lines, stressed conditions, etc.) for which experimental data is unavailable

FIG. 9 shows an example to highlight how the process described in FIG. 8 can be applied to identify biomolecular condensates. Data on the genetic variations that are present in disease population is used in conjunction with the predictive models of condensate behavior to identify genetic alterations that result in abnormal biomolecular condensation landscape. The process allows identifying targets with genetic evidence and those that have biomarker-based subpopulations. Shown in the highlighted box: genetic alterations can lead to aberrant condensation by multiple routes. Examples include, without limitation, (i) genetic alterations that alter the concentration of the target (increased or decreased expression level via altered rate of translation or degradation), (ii) genetic alterations that affect the phase behavior of the sequence (e.g. mutated sequence having an altered condensation propensity, mutation leading to a modified post-translational state) or (Hi) genetic alterations resulting in the environment changing in a manner where the condensation of the targets is altered (e.g. changes in the concentration of the modulator molecule).

FIG. 10 shows how saturation mutagenesis profiling allows identification of regions of a protein sequence that are the most sensitive to mutations. All residues of the sequence are mutated and run through a predictor that links sequence to a phase-separation-propensity score. Variations in the scores are used to construct a profile highlighting the importance of the different regions for the phase separation process, such as the one shown on bottom right using CTNNB1 as the example. Highlighted regions are important for the phase separation process. Mutations occurring in a patient population of interest can then be overlaid with this profile to identify cases where mutations are enriched into regions that are important for the phase separation process.

FIG. 11 shows an example of how the strategy outlined in FIGS. 9-10 is reduced to practice to identify condensate targets across a variety of patient cohorts. Genetic data across the coding genome is analyzed for the cohort of interest to identify cases where mutations are enriched into the regions that are important for the phase separation process. The process identifies CTNNB1 as a condensate target of interest as mutations are enriched into regions important for the phase separation process. Optionally, it may be required that these mutations are not present or are present much less frequently among health volunteers.

FIG. 12 shows how data from cellular models can be further integrated to further in silico validate the condensate target. As an example, CTNNB1 shows high dependency in colorectal cell lines with the dependency being elevated upon mutations within the region that are important for the phase separation process as outlined in FIG. 10.

FIG. 13 shows an exemplary target from the in-silico target identification pipeline undergoing experimental validation. The protein undergoes phase separation in a purified form (left). It also forms condensates in the identified disease context (colorectal cell lines with N-terminal CTTNB1 mutations).

DEFINITIONS

In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; and (Hi) the terms “including” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps.

As used herein, the terms “about” and “approximately” refer to a value that is within 10% above or below the value being described. For example, the term “about 5 nM” indicates a range of from 4.5 to 5.5 nM.

As used herein, “comprising,” “having,” and “including” and grammatical equivalents thereof, are intended to be equivalent in meaning and to be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an object “comprising” components X, Y, and Z can consist of (i.e ., contain only) components X, Y, and Z, or can contain not only components X, Y, and Z, but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiment of “consisting essentially of” or “consisting of.”

As used herein, a “cellular composition” is a composition comprising at least one cell. Exemplary compositions include a tissue sample or cultured cells.

As used herein, “condensate” means a non-membrane-encapsulated compartment formed by LLPS of one or more of proteins and/or other macromolecules (including all stages of LLPS).

As used herein, the term “macromolecule” refers to a protein, polypeptide, RNA or DNA.

As used herein, “protein” and “polypeptide” refer to a polymer of amino acid residues and are not limited to a minimum length. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-translational modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.

As used herein, “RNA” and “DNA” refer to polymeric form of nucleotides of any length, including ribonucleotides and deoxyribonucleotides. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Thus, this term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, mRNA, 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 backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. The backbone of the polynucleotide can comprise repeating units, such as N-(2-aminoethyl)-glycine, linked by peptide bonds (e.g., peptide nucleic acid). Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P — NH2) or a mixed phosphoramidate-phosphodiester oligomer.

As used herein, “target macromolecule” refers to a macromolecule that can be found in or on a condensate under physiological or pathological conditions.

As used herein, “label” and “barcode” refer to a detectable tag that can be attached, bound or a component of the compound of interest, or the target macromolecule. Exemplary tags include fluorescent dyes, epitope tags, and radiolabels.

As used herein, “c_Sat” refers to concentration of the macromolecule at which the molecule transitions from a homogenous single-phase system to a multi-phase system, such as a two-phase system that includes bimolecular condensates

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 dictate 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 disclosure, 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 disclosure.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides, in some aspects, the discovery of therapeutic agents for the treatment of condensate-based diseases. Aberrant conversion of proteins between the native, amyloid and droplet states is responsible for a number of condensate-based diseases, such as amyolateral sclerosis (ALS), Alzheimer’s, and Huntington’s etc. This invention is based, at least in part, on the inventor’s unique understanding of various screening methods and the application of such methods to understand the effects of agents for the treatment of diseases. Previous methods of screening compounds focused on single step methods to identify compounds, or on a compound-by-compound base to identify compounds that selectively modulate a condensate or its components without disruption of the entire condensate. The methods disclosed herein enable techniques for the screening a plurality of compounds to identify compounds that modulate the liquid-liquid phase separation (LLPS) of the target macromolecule in one or more phases, and/or the multistep screening of compounds to identify condensate modulators.

In the first aspect, the disclosure provides a method of identifying condensate modulators, the method comprising contacting a condensate modulator with a composition comprising a target macromolecule and measuring one or more phase transition characteristics of the condensate, the method comprising: (a) producing a stream of microdroplets, wherein each of said microdroplets comprises a compound and the target macromolecule, wherein the concentration of the compound varies among the plurality of microdroplets; (b) determining, within each of the microdroplets, one or more phase transition characteristics of the condensate; and (c) determining whether the compound modulates one or more phase transition characteristics of the condensate, wherein: the determining is performed by comparing the concentration of the compound at which the one or more phase transition characteristics change the one or more phase transition characteristics change as compared to a concentration of a reference compound at which the one or more phase transition characteristics change the one or more phase transition characteristics change.

In another aspect, the disclosure provides a method of identifying selective condensate modulators, the method comprising contacting a condensate modulator with a composition comprising a target macromolecule and a non-target macromolecule, and measuring one or more phase transition characteristics of the condensate. In another aspect, the disclosure provides a method of identifying selective condensate modulators, the method comprising contacting a condensate modulator with a composition comprising a target macromolecule and a non-target macromolecule, and measuring one or more phase transition characteristics of the condensate in a cell. In another aspect, the disclosure provides a method of identifying selective condensate modulators, the method comprising contacting a condensate modulator with a composition comprising a target macromolecule and a non-target macromolecule, and measuring one or more phase transition characteristics of the condensate in vitro and/or in a cell.

In another aspect, the disclosure provides a method for determining whether a compound modulates one or more phase transition characteristics of a condensate in a cell, the method comprising: (a’) introducing, into a cell, the compound identified as one that modulates one or more phase transition characteristics of the condensate in vitro, wherein the cell comprises the target macromolecule; and (b’) assessing whether the compound modulates one or more phase transition characteristics of the condensate in the cell, wherein: the determining is by comparing the concentration of the compound at which the one or more phase transition characteristics change in the cell, as compared to a concentration of a reference compound at which the one or more phase transition characteristics change in the cell.

In another aspect, the disclosure provides a method for screening a plurality of compounds to identify condensate modulators that inhibit or promote LLPS of target macromolecules, the method comprising: (a) producing a stream of microdroplets, wherein each of said microdroplets comprises a compound and the target macromolecule, wherein the concentration of the compound varies among the plurality of microdroplets; (b) determining, within each of the microdroplets, whether the target macromolecule has undergone LLPS, thereby identifying the concentration of the compound at which the target macromolecule undergoes LLPS; and (c) determining whether the compound promotes or inhibits LLPS, wherein: the determining is performed by comparing the concentration of the compound at which the target macromolecule undergoes LLPS as compared to a concentration of a reference compound at which the target macromolecule undergoes LLPS, the compound promotes LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is less than the concentration of the reference compound at which the target macromolecule undergoes LLPS, and the compound inhibits LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is greater than the concentration of the reference compound at which the target macromolecule undergoes LLPS.

In another aspect, the disclosure provides a method of determining whether a compound selectively inhibits or promotes LLPS of a target macromolecules, the method comprising: (a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, a non-target macromolecule and wherein the concentration of the compound varies among the plurality of microdroplets; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-tar et) , wherein fluorescence of each non-target fluorescent dye (FDnon-tar et) facilitates detection of one non-target macromolecule, (b) determining, within each of the microdroplets, whether the target macromolecule has undergone LLPS, and whether the non-target macromolecule has undergone LLPS, thereby identifying the concentration of the compound at which the target macromolecules undergo LLPS and the concentration of the compound at which the non- target macromolecule undergoes LLPS; (c) determining whether the compound selectively promotes or inhibits LLPS of the target macromolecules, wherein: the determining is by comparing the concentration of the compound at which the target macromolecules undergo LLPS as compared to a concentration of at which the non-target macromolecule undergoes LLPS, the compound promotes LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is less than the concentration of the compound at which the non-target macromolecule undergoes LLPS, and the compound inhibits LLPS when the concentration of the compound at which the target macromolecules undergo LLPS is greater than the concentration of the compound at which the nontarget macromolecule undergoes LLPS, (a’) introducing, into a cell, the compound identified in (c) as one that selectively promotes or inhibits LLPS of the target macromolecules in vitro, wherein the cell comprises the target macromolecule and the non-target macromolecule; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non- covalently bound to a fluorescent dye (FDnon-tar et), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, and (b’) assessing whether the compound selectively promotes or inhibits LLPS of the target macromolecule in the cell, wherein: the determining is by comparing the concentration of the compound at which the target macromolecules undergo LLPS in the cell, as compared to a concentration of the compound at which the non-target macromolecule undergoes LLPS in the cell, the compound promotes LLPS in the cell when the concentration of the compound at which target macromolecules undergoes LLPS in the cell is less than the concentration of the compound at which the non-target macromolecule undergoes LLPS in the cell, and the compound inhibits LLPS in the cell when the concentration of the compound at which the target macromolecules undergo LLPS in the cell is greater than the concentration of the compound at which the non-target macromolecule undergoes LLPS in the cell.

In another aspect, the disclosure provides a method of identifying a compound useful for treating a disease in an individual in need thereof, wherein the LLPS of the target macromolecule is associated with the disease, and wherein the compound is identified as one that inhibits the LLPS of the target macromolecule, thereby identifying a compound useful for treating the disease. In some embodiments, the method comprising administering a therapeutically effective amount of the compound to an individual diagnosed as having the disease.

In another aspect, the disclosure provides a target macromolecule, the method comprising: (a) screening a multimodal data set from a plurality of human biological samples to identify a plurality of genetic variations that distinguish a human disease state from a human healthy state;(b) screening a multimodal data set from a plurality of in vitro disease relevant cell line models to identify a plurality of genetic variations that distinguish a disease state from a healthy state; (c) analyzing the plurality of genetic variations to identify one or a subset of genetic variations that lead to a change in concentration of the target macromolecule, a chemical alternation of the target macromolecule, and/or a change in the endogenous environment of the target macromolecule; (a’) determining that the change in concentration, the chemical alteration and/or change in endogenous environment of the target leads to aberrant condensation behavior in the disease-associated model; and (b’) determining that the change described in (a’) does not lead to an aberrant condensation behavior in biological samples from healthy volunteers or non-diseased cell models.

Methods of Screening and Identifying Compounds

In some aspects of the application, methods for screening a plurality of compounds to identify modulators of one or more phase transition characteristics of a condensate and applications thereof. In some aspects, provided herein are methods of high-throughput screening and/or identifying compounds that inhibit or promote phase transition of the target macromolecule. In some aspects of the application, methods of identifying compounds that selectively inhibit or promote phase transition of the target macromolecule are provided. In some aspects of the application, methods of identifying compounds that modulate one or more phase transition characteristics of a condensate are provided. In some aspects, provided herein are methods of screening for compounds that modulate one or more phase transition characteristics of a condensate. In some aspects, provided herein are methods of high- throughput screening and/or identifying compounds that modulate one or more phase transition characteristics of a condensate. In some aspects of the application, methods of identifying compounds that selectively modulate one or more phase transition characteristics of a condensate are provided.

In some aspects of the application, methods of identifying compounds that inhibit or promote LLPS of the target macromolecule are provided. In some aspects, provided herein are methods of screening for compounds that inhibit or promote LLPS of the target macromolecule. In some aspects, provided herein are methods of high- throughput screening and/or identifying compounds that inhibit or promote LLPS of the target macromolecule. In some aspects of the application, methods of identifying compounds that selectively inhibit or promote LLPS of the target macromolecule are provided.

Those skilled in the art will recognize that, in view of the provided description, several embodiments are possible within the scope and spirit of the disclosure of this application.

In one aspect, provided herein are methods for screening (such as high throughput screening) to identify modulators of one or more phase transition characteristics of a condensate, the method comprising: (a) producing a stream of microdroplets, wherein each of said microdroplets comprises a compound and the target macromolecule, wherein the concentration of the compound varies among the plurality of microdroplets; (b) determining, within each of the microdroplets, one or more phase transition characteristics of the condensate; and (c) determining whether the compound modulates one or more phase transition characteristics of the condensate, wherein: the determining is performed by comparing the concentration of the compound at which the one or more phase transition characteristics change the one or more phase transition characteristics change as compared to a concentration of a reference compound at which the one or more phase transition characteristics change the one or more phase transition characteristics change.

In another aspect, provided herein are methods of identifying compounds that selectively inhibit or promote phase transition of target macromolecules in the presence of non-target macromolecule in vitro and/or in a cell. In another aspect, the disclosure provides a method of identifying compounds that selectively modulate phase transition of target macromolecules in the presence of non-target macromolecule in vitro and/or in a cell.

In another aspect, provided herein are methods for identifying a compound modulates one or more phase transition characteristics of a condensate in a cell, the method comprising: (a’) introducing, into a cell, the compound identified as one that modulates one or more phase transition characteristics of the condensate in vitro, wherein the cell comprises the target macromolecule; and (b’) assessing whether the compound modulates one or more phase transition characteristics of the condensate in the cell, wherein: the determining is by comparing the concentration of the compound at which the one or more phase transition characteristics change in the cell, as compared to a concentration of a reference compound at which the one or more phase transition characteristics change in the cell.

In one aspect, provided herein are methods of screening (such as high throughput screening) for compounds that inhibit or promote LLPS of the target macromolecule, the method comprising assessing a plurality of compounds in a screen using any of the methods provided herein. In some embodiments, the method of screening (such as high throughput screening) for a compound or compounds that inhibit or promote LLPS of the target macromolecule, comprises (a) producing a stream of microdroplets consisting of various concentrations of the compound, (b) determining, within each of the microdroplets, whether the target macromolecule has undergone LLPS, thereby identifying the concentration of the compound at which the target macromolecule undergoes LLPS; and (c) determining whether the compound promotes or inhibits LLPS, wherein: the determining is performed by comparing the concentration of the compound at which the target macromolecule undergoes LLPS as compared to a concentration of a reference compound at which the target macromolecule undergoes LLPS, the compound promotes LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is less than the concentration of the reference compound at which the target macromolecule undergoes LLPS, and the compound inhibits LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is greater than the concentration of the reference compound at which the target macromolecule undergoes LLPS.

In some embodiments, provided herein are methods of screening (such as high throughput screening) for compounds that selectively inhibit or promote LLPS of the target macromolecule, the method comprising assessing a plurality of compounds in a screen using any of the methods provided herein. In some embodiments, the method of screening (such as high throughput screening) for a compound or compounds that inhibit or promote LLPS of the target macromolecule, comprises (a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, and wherein the concentration of the compound varies among the plurality of microdroplets; (b) determining, within each of the microdroplets, whether the target macromolecule has undergone LLPS, thereby identifying the concentration of the compound at which the target macromolecules undergo LLPS and the concentration of the compound at which the nontarget macromolecule undergoes LLPS; (c) determining whether the compound selectively promotes or inhibits LLPS of the target macromolecules, wherein: the determining is by comparing the concentration of the compound at which the target macromolecules undergo LLPS as compared to the concentration of a reference compound at which the target macromolecule undergoes LLPS; then (a’) introducing, into a cell, the compound identified in (c) as one that selectively promotes or inhibits LLPS of the target macromolecules in vitro, wherein the cell comprises the target macromolecule and the non-target macromolecule; and (b’) assessing whether the compound selectively promotes or inhibits LLPS of the target macromolecule in the cell, wherein: the determining is by comparing the concentration of the compound at which the target macromolecules undergo LLPS in the cell, as compared to a concentration of the compound at which the non-target macromolecule undergoes LLPS in the cell. In some embodiments, provided herein are methods of screening (such as high throughput screening) for compounds that selectively inhibit or promote LLPS of the target macromolecule, the method comprising assessing a plurality of compounds in a screen using any of the methods provided herein. In some embodiments, the method of screening (such as high throughput screening) for a compound or compounds that inhibit or promote LLPS of the target macromolecule, comprises (a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, a non-target macromolecule, and wherein the concentration of the compound varies among the plurality of microdroplets; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-tar et) , wherein fluorescence of each non-target fluorescent dye (FDnon-tar et) facilitates detection of one non-target macromolecule, (b) determining, within each of the microdroplets, whether the target macromolecule has undergone LLPS, and whether the non-target macromolecule has undergone LLPS, thereby identifying the concentration of the compound at which the target macromolecules undergo LLPS and the concentration of the compound at which the non- target macromolecule undergoes LLPS; (c) determining whether the compound selectively promotes or inhibits LLPS of the target macromolecules, wherein: the determining is by comparing the concentration of the compound at which the target macromolecules undergo LLPS as compared to a concentration of at which the non-target macromolecule undergoes LLPS, then (a’) introducing, into a cell, the compound identified in (c) as one that selectively promotes or inhibits LLPS of the target macromolecules in vitro, wherein the cell comprises the target macromolecule and the non-target macromolecule; and (b’) assessing whether the compound selectively promotes or inhibits LLPS of the target macromolecule in the cell, wherein: the determining is by comparing the concentration of the compound at which the target macromolecules undergo LLPS in the cell, as compared to a concentration of the compound at which the non-target macromolecule undergoes LLPS in the cell.

In some embodiments, the method of screening comprises (a) producing a composition that comprises (i) a compound, (ii) a target macromolecules wherein the target macromolecules is a protein (iii) a non-target macromolecule wherein the non-target macromolecule is RNA, (b) measuring the relative concentrations of the compound, and the relative concentration of the target macromolecule, and (c) determining whether the compound selectively promotes or inhibits LLPS of the target macromolecules, wherein: the determining is by comparing the concentration of the compound at which the target macromolecules undergo LLPS as compared to a concentration of at which the non-target macromolecule undergoes LLPS.

In some embodiments, the microdroplet is mounted on a microfluidic chip. In some embodiments, the microdroplet comprises various concentrations of the compound, and various concentrations of the target macromolecule. In some embodiments, the microdroplet comprises a trigger of LLPS or phase transition. In some embodiments, the microdroplet comprises buffer, salt solution and a trigger of LLPS or phase transition. In some embodiments, the microdroplet comprises a dye, e.g., Alexa 546 or Alexa 647. In some embodiments, the temperature of the microdroplets is varied by controlling the temperature of a channel in which the microdroplets flow. In some embodiments, the concentration of the compound, and the target macromolecule in the microdroplet is controlled by the flowrate. In some embodiments, the stream of microdroplets is a continuous stream.

In some embodiments, the plurality of compounds comprises at least 100, 250, 500, 1000, 2000, 3000, 5000, 10000, or more different compounds. In some embodiments, the plurality of compounds is allowed to be in contact with the composition in separate reactions (e.g., separate vessels or wells). In some embodiments, the plurality of compounds is allowed to be in contact with the composition in separate reactions simultaneously.

In some embodiments, the methods described herein comprise producing a stream of microdroplets consisting of the compound and the target macromolecule that has undergone LLPS or phase transition, partially or completely, or a microdroplet capable or undergoing LLPS or phase transition. One of ordinary skill in the art will readily recognize that biological processes, including the state of a condensate and components thereof, are dynamic. The methods described herein thus encompass producing a stream of microdroplets of the compound and the target macromolecule at any point in the life cycle of the one or more phases. For examples, the methods encompass producing a stream of microdroplets of the compound and the target macromolecule are present in any quantity, including being absent, are undergoing a morphological change, such as a change in size of liquidity, or are changing in composition.

In some embodiments, the LLPS or phase transition occurs simultaneously after producing the stream of microdroplets. In some embodiments, the microdroplet is subject to a trigger prior to determining the LLPS or phase transition of the target macromolecule. In some embodiments, the microdroplet comprises one or more phases, and additional LLPS or phase transition occurs simultaneously with contacting the microdroplet with the microfluidic chip. In some embodiments, the microdroplet comprises one or more phases, and additional LLPS or phase transition occurs after contacting the microdroplet with the microfluidic chip. In some embodiments, the microdroplet comprises one or more phases, and additional LLPS or phase transition occurs simultaneously with and after contacting the microdroplet with the microfluidic chip.

In some embodiments, the target macromolecule undergoes LLPS or phase transition prior to contact with the microfluidic chip. In some embodiments, the method further comprises subjecting the microdroplet to a trigger prior to contacting the microfluidic chip. In some embodiments, the method further comprises contacting the microfluidic chip with a microdroplet comprising the target macromolecule that has undergone LLPS or phase transition, or with a microdroplet that is capable or undergoing LLPS or phase transition. In some embodiments, the LLPS or phase transition occurs simultaneously after producing the stream of microdroplets.

In some embodiments, the methods described herein comprise contacting the microdroplet comprising one or more phases, or a microdroplet capable or undergoing LLPS or phase transition with a trigger of LLPS or phase transition. In some embodiments, the microdroplet contains 0.5-6% of the trigger of LLPS or phase transition. In some embodiments, the trigger of LLPS or phase transition is PEG10k or a salt, such as NaCI or KCI, among others. In some embodiments, the trigger of LLPS or phase transition is a biological mixture, such as a cell lysate.

As described herein, the methods include producing a stream of microdroplets of the compound and the target macromolecule wherein (a) the target macromolecule has not undergone LLPS; and/or (b) the target macromolecule undergoes LLPS simultaneously with and/or after producing the stream of microdroplet. In some embodiments, the methods include producing a stream of microdroplets of the compound and the target macromolecule wherein (a) the target macromolecule has not undergone LLPS; and/or (b) is capable of undergoing LLPS, wherein the target macromolecule undergoes LLPS with and/or after producing the stream of microdroplets. In some embodiments, the methods described herein include producing a stream of microdroplets of the compound and the target macromolecule undergoes LLPS or a cellular composition capable of undergoing LLPS.

In some embodiments, the conditions to form the condensate comprises the addition of a trigger or the exposure of the cellular composition to a physical stressor. In some embodiments, the method comprises subjecting the microdroplet to any one or more of the following: (a) an oxidative stressor, (b) a mitochondrial electron transport chain inhibitor, (c) a heat stressor, (d) an osmotic stressor, (e) a hyperosmotic stressor (f) glycolysis inhibitor and (g) a salt solution. In some embodiments, the trigger is sodium arsenate, sorbitol, rotenone, 6-deoxyglucose in the absence of glucose, Actinomycin D, or Adenosine dialdehyde (AdOx). In some embodiments, the physical stressor is a heat stressor exposing the cellular composition to a temperature of 40-45°C, such as 43°C. In some embodiments, the physical stressor is an aging condition, e.g., incubation, shaking, and/or heat.

In some embodiments, the reference is a microdroplet that was treated with a reference compound. In some embodiments, the reference is an experimental control. In some embodiments, the reference is a microdroplet that does not comprise the compound. In some embodiments, the reference is a microdroplet that was not treated with a trigger or physical stressor. In some embodiments, the reference is prepared in a manner such that a meaningful result can be assessed for the compound. For example, in some embodiments, the reference is a microdroplet, wherein the microdroplet is prepared in a similar manner as the microdroplet comprising the compound, except the reference microdroplet does not comprise the compound. In some embodiments, the reference is a microdroplet that comprises a reference compound, such as a trigger of LLPS or phase transition or negative control compound.

In some embodiments, the positive reference microdroplet comprises a trigger of LLPS or phase transition, the target macromolecule, non-target macromolecules, a phase-separation trigger, and buffer. In some embodiments, the trigger of LLPS or phase separation is 5% w/v 1 ,6-hexanediol. In some embodiments, the positive reference microdroplet comprises a trigger of LLPS or phase separation, the target macromolecule, and buffer. In some embodiments, the positive reference microdroplet comprises a trigger of LLPS or phase separation, the target macromolecule, a phaseseparation trigger, and buffer. In some embodiments, the positive reference microdroplet comprises a trigger of LLPS or phase separation, the target macromolecule, and buffer. In some embodiments, the positive reference microdroplet comprises a trigger of LLPS or phase separation, and the target macromolecule.

In some embodiments, the negative reference microdroplet comprises a negative control compound, the target macromolecule, non-target macromolecules, and buffer. In some embodiments, the negative control compound is 1% v/v DMSO. In some embodiments, the negative reference microdroplet comprises a negative control compound, the target macromolecule, non-target macromolecules, and buffer. In some embodiments, the negative reference microdroplet comprises a negative control compound, and the target macromolecule. In some embodiments, the negative reference microdroplet comprises the target macromolecule.

In some embodiments, the concentration of the compound is between about 1 .2 pM and 100 pM. In some embodiments, the trigger of LLPS or phase separation is between about 0.5 - 6% w/v PEG 10k in each microdroplet. Phase Transition

In some embodiments, measuring the phase transition of the target macromolecule is based on any one or more of the following: (i) number of phase separated target macromolecules; (ii) size of the one or more phases; (iii) location of the one or more phases; (iv) distribution of one or more phases; (v) surface area of the one or more phases; (vi) composition of the one or more phases, (vii) liquidity of the one or more phases; (viii) solidification of the one or more phases; (ix) dissolution of the one or more phases; (x) location of the target macromolecule; (xi) partitioning of the target macromolecule; and (xii) aggregation of the target macromolecule.

In some embodiments, the measuring is performed within about 60 days of the producing of the stream of microdroplets, such as within about 35 days, about 28 days, about 21 days, about 14 days, about 10 days, about 7 days, about 5 days, about 3 days, about 2 days, about 1 day, about 12 hours, about 5 hours, about 2 hours, about 1 hour, about 45 minutes, about 30 minutes, about 15 minutes, about 5 minutes, about 1 minute, or about 30 seconds. In some embodiments, the determining is performed after about 5 seconds of the producing of the stream of microdroplets, such as, after about 15 seconds, about 30 seconds, about 1 minute, about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 5 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 5 days, about 7 days, about 10 days, about 14 days, about 21 days, about 28 days, about 35 days, or about 60 days.

In some embodiments, the method further comprises repeating the measuring step of the method. For example, in some embodiments, the methods comprise repeating the measuring step of the method at least about any of 2, 3, 4, 5, 10, or more times. In some embodiment, the methods comprise repeating the measuring step of the method on at least 1 , 2, 3, 4, 5, 10 or more microfluidic chips.

In some embodiments, the measuring step of the method is repeated after an interval of time, such as about 30 seconds, about 1 minute, about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 5 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 5 days, about 7 days, about 10 days, about 14 days, about 21 days, about 28 days, about 35 days, about 60 days, or more. In some embodiments, the method further comprises comparing the phase transition over time, such as comparing the number of condensates with one day between measurements.

In some embodiments, the microdroplets, and condensates are visualized by microcopy, including for example, stereoscopic microscopy, brightfield microscopy, polarizing microscopy, phase contrast microscopy, differential interference contrast microscopy, fluorescence microscopy, total internal reflection fluorescence microscopy, confocal microscopy, or multiphoton excitation microscopy. In some embodiment, the visualization is performed continuously on the stream of microdroplets. In some embodiments, the microdroplets and condensates are visualized by multicolor epifluorescence microscope. In some embodiments the multi-color epifluorescence microscope excites the microdroplets and detects a response. In some embodiments, the multi-color epifluorescence microscope excites and detects a response on multiple different fluorescence channels. In some embodiments, the multiple fluorescence channels are 470nm, 555nm and 640 nm. In some embodiments, the excitation is light via a laser diode. In some embodiments, the laser diode emits a wavelength of 470nm, 555nm, or 640 nm. In some embodiments, the relative concentrations of the compounds and each of the target macromolecule are determined based on the respective responses of each target macromolecules to the excitation. In some embodiments, the relative concentration of the compounds is determined from the fluorescent intensity of Alexa 546 or Alexa 647 dyes. In some embodiments, the relative concentration of each of the target macromolecules comprise a different fluorophore which emits a specific wavelength in response to excitation.

LLPS

In some embodiments, measuring the LLPS of the target macromolecule is based on any one or more of the following: (i) number of phase separated target macromolecules; (ii) size of the one or more phases; (iii) location of the one or more phases; (iv) distribution of one or more phases; (v) surface area of the one or more phases; (vi) composition of the one or more phases, (vii) liquidity of the one or more phases; (viii) solidification of the one or more phases; (ix) dissolution of the one or more phases; (x) location of the target macromolecule; (xi) partitioning of the target macromolecule; and (xii) aggregation of the target macromolecule.

In some embodiments, the method further comprises repeating the measuring step of the method. For example, in some embodiments, the methods comprise repeating the measuring step of the method at least about any of 2, 3, 4, 5, 10, or more times. In some embodiment, the methods comprise repeating the measuring step of the method on at least 1 , 2, 3, 4, 5, 10 or more microfluidic chips. In some embodiments, the measuring step of the method is repeated after an interval of time, such as about 30 seconds, about 1 minute, about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 5 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 5 days, about 7 days, about 10 days, about 14 days, about 21 days, about 28 days, about 35 days, about 60 days, or more. In some embodiments, the method further comprises comparing the LLPS over time, such as comparing the number of condensates with one day between measurements.

Fluorescent Dyes

In some embodiments, the compounds, condensates and/or target macromolecules may be labeled, for example, labeled with a fluorophore. In some embodiments, the compounds, condensates and/or the target macromolecule can be conjugated to a detectable label, such as a fluorescent molecule, epitope tag, or radiolabel. In some embodiments, the compound, the one or more target macromolecule, and condensate comprises a fluorescent dye, such as a fluorescent protein, e.g., GYP, CFP, or RFP, etc. In some embodiments, the method comprises labeling at least a portion of the microdroplet. In some embodiments, the label is a labeled binding molecule, such as an antibody or biotin-binding protein. In some embodiments, the label is a stain, such as a stain specific to an organelle.

In some embodiments, the fluorescent dye may be green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, phycoerythrin, allophycocyanin, hoescht, ”,6-diamidino-2-phenylindole (DAPI), propidium iodide, fluorescein, coumarin, rhodamine, tetramethylrhoadmine, or cyanine. In some embodiments, the epitope tag may be maltose-binding protein, glutathione-S-transferase, a poly-histidine tag, a FLAG-tag, a myc-tag, human influenza hemagglutinin (HA) tag, biotin, or streptavidin. In some embodiments, the epitope tag (e.g., biotin, avidin, FLAG tag, HA tag) can later be detected by treatment with a complementary tag (e.g., avidin, biotin, anti-FLAG antibody, anti-HA antibody, respectively). In some embodiments, epitope tag may be maltose-binding protein, glutathione-S-transferase, a poly-histidine tag, a FLAG-tag, a myc-tag, human influenza hemagglutinin (HA) tag, biotin, or streptavidin. In some embodiments, the tag can be maltose, glutathione, a nickel-containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti-HA antibody, biotin, or streptavidin. In some embodiments, the tag can green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, phycoerythrin, allophycocyanin, hoescht, ”,6-diamidino-2-phenylindole (DAPI), propidium iodide, fluorescein, coumarin, rhodamine, tetramethylrhoadmine, and cyanine.

In some embodiments, the fluorescent dye may be selected from 7-nitrobenz-2-oxa-1 ,3- diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUE™ dye), 5- (dimethylamino)naphthalene-l -sulfonyl (Dansyl), pyrene, 7-amino-3-{[(2,5-dioxopyrrolidin-1 -yl)oxy]-2- oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350™), 6,8-difluoro-7- hydroxy-4-methylcoumarin (MARINA BLUE™ dye), N-(2-aminoethyl)-4-{5-[4-(dimethylamino)phenyl]- 1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYL™ dye), 2,3,5,6-Tetramethyl-1 H,7H-pyrazolo[1 ,2- a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4-(ethylamino)naphthalen-2- yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUE™ dye), tris(N,N- diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrol idin-1 -yl)oxy]carbonyl}piperidin- 1 -yl)-2- oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405™), N,N-diethylethanaminium [9-{6-[(2,5- dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H- benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430™), 1 -[({4-[(7-nitro-2,1 ,3-benzoxadiazol- 4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSY™ dye), fluorescein, 2-(6-amino-3-iminio-4,5- disulfonato-3H-xanthen-9-yl)-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488™), ’',”-Difluoro-”,”-dihydroxy-3H-spiro[isobenzofuran-1 ,”-xanthen]-3-one (OREGON GREEN™ 488), 1 ,3,5,7,8-pentamethyl-4,4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPY™ 493/503), rhodamine green, and rhodamine red. For example, in some embodiments, the compound is 2-[N-(7- nitrobenz-2-oxa-1 ,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG).

In some embodiments, the fluorescent dye is selected from 7-nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUE™ dye), 5- (dimethylamino)naphthalene-l -sulfonyl (Dansyl), pyrene, 7-amino-3-{[(2,5-dioxopyrrolidin-1 -yl)oxy]-2- oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350™), 6,8-difluoro-7- hydroxy-4-methylcoumarin (MARINA BLUE™ dye), N-(2-aminoethyl)-4-{5-[4-(dimethylamino)phenyl]- 1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYL™ dye), 2,3,5,6-Tetramethyl-1 H,7H-pyrazolo[1 ,2- a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4-(ethylamino)naphthalen-2- yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUE™ dye), tris(N,N- diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrol idin-1 -yl)oxy]carbonyl}piperidin- 1 -yl)-2- oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405™), N,N-diethylethanaminium [9-{6-[(2,5- dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H- benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430™), 1 -[({4-[(7-nitro-2,1 ,3-benzoxadiazol- 4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSY™ dye), fluorescein, 2-(6-amino-3-iminio-4,5- disulfonato-3H-xanthen-9-yl)-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488™), ’',”-Difluoro-”,”-dihydroxy-3H-spiro[isobenzofuran-1 ,”-xanthen]-3-one (OREGON GREEN™ 488), 1 ,3,5,7,8-pentamethyl-4,4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPY™ 493/503), rhodamine green, 13-[2-carboxy-3,4,6-trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 -yl)oxy-6- oxohexyl]amino]-2-oxoethyl]sulfanylphenyl]-7,7,9,17,19,19-hexamethyl-2-oxa-6,20- diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa-1 (14),3,5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546™), 2-[5-[3,3-dimethyl-5-sulfo-1 -(3-sulfopropyl)indol-1 -ium-2-yl]penta-2,4- dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5- sulfonic acid (ALEXA FLUOR 647™), and rhodamine red; and/or wherein one or more of the fluorescent dye is a fluorescent protein selected from GFP, mNeonGreen, mRFP, mCherry, and mPlum.

In some embodiments, the method comprises constructing a phase diagram. In some embodiments, the phase diagram is constructed on a drop-by-drop basis. In some embodiments, the phase of the target macromolecules is determined based on characteristics of the image indicative of particular phases.

The LLPS of target macromolecules can be determined for a portion or all of the microdroplet. Accordingly, in some embodiments, the method comprises determining the LLPS of the target macromolecule in a portion of the microdroplet. In some embodiments, the method comprises determining the LLPS of the target macromolecule in the entire microdroplet.

Compounds

“Compound” used herein refers to any agent. In some embodiments, the compound is a small molecule, a polypeptide, a lipid, or a nucleic acid. In some embodiments, the compound is an approved compound, such as a compound approved for medical treatment by the United States Food and Drug Administration. In some embodiments, the compound is a novel compound. In some embodiments, the compound is charged. In some embodiments, the compound is hydrophobic. In some embodiments, the compound is hydrophilic. In some embodiments, the compound is a small molecule. In some embodiments, the small molecule is an alkaloid, a glycoside, a phenazine, a phenol, a polyketide, a terpene, or a tetrapyrrole. In some embodiments, the compound is an antibody. In some embodiments, the compound is a nucleic acid. In some embodiments, the compound is RNA, such as a siRNA, miRNA, or mRNA. In some embodiments, the compound is a non-naturally occurring compound. In some embodiments, the compound is a naturally occurring compound. When a plurality of candidate compounds is used for screening, the plurality of candidate compounds can be of the same type or of different types.

In some embodiments, the compound library comprises from 10 to 100,000 compounds, or more. In some embodiments, the concentration of the compound at which the target macromolecule undergoes LLPS is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet, wherein the positive reference microdroplet comprises the target macromolecule and a trigger of LLPS, and the negative reference microdroplet comprises the target macromolecule. In some embodiments, the concentration of the compound at which the target macromolecule undergo LLPS is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet, wherein the positive reference microdroplet comprises the target macromolecule and a trigger of LLPS, and the negative reference microdroplet comprises the target macromolecule.

In some embodiments, the compound is capable of selectively interacting non-covalently with a macromolecule (particularly a protein or nucleic acid) under conditions prevailing in a live cell, wherein said compound, and said biomolecule form a complex having a dissociation constant Kd of 10-4 mol/l or less.

In some embodiments, the compound is capable of selectively interacting non-covalently with a macromolecule (particularly a protein or nucleic acid). In some embodiments, the compound is capable of selectively interacting non-covalently with a macromolecule (particularly a protein) that is bound non-covalently to another macromolecule (particularly a nucleic acid or RNA). In some embodiments, the compound is capable of selectively interacting non-covalently with a macromolecule (particularly a protein) that is bound covalently to another macromolecule (particularly a nucleic acid or RNA). In some embodiments, the compound is capable of selectively interacting non-covalently with a macromolecule (particularly a nucleic acid) that is bound non-covalently to another macromolecule (particularly a target protein). In some embodiments, the compound is capable of selectively interacting non-covalently with a macromolecule (particularly a nucleic acid or RNA) that is bound covalently to another macromolecule (particularly a target protein). Protein-RNA conjugate assay

In one aspect, the disclosure provides a high throughput method for screening a plurality of compounds to identify compounds that modulate the LLPS of the target macromolecule in one or more phases, wherein the method comprises a protein-RNA conjugate assay to identify compounds that modulate the LLPS of protein-RNA binary interactions of the target macromolecule in the one or more phases.

In some embodiments, the protein interacts with the RNA. In some embodiments, the compound modulates the protein-RNA binary interaction. In some embodiments, the compound does not modulate the protein-RNA binary interaction. In some embodiments, the compound increases the protein-RNA binary interaction. In some embodiments, the compound decreases the protein-RNA binary interaction.

Cells and Condensates

In some embodiments, the cellular composition comprises a microorganism or an animal cell. In some embodiments, the cellular composition comprises a human cell. In some embodiments, the cellular composition comprises a neuron. In some embodiments, the cellular composition comprises a cancer cell. In some embodiments, the cellular composition comprises a cell that is or is derived from induced pluripotent stem cells (iPS cells), HeLa cells, or HEK293 cells. In some embodiments, the cellular composition comprises a condensate that is dysregulated. In some embodiments, the cellular composition comprises a cell comprising a mutation associated with a disease. In some embodiments, the cellular composition comprises a cell having one or more features of a neurodegenerative or proliferative disease. In some embodiments, the cellular composition comprises a cell expressing a protein that is labeled with a fluorescent protein. In some embodiments, the protein is a protein known to concentrate in a condensate. In some embodiments, the target macromolecule is labeled, such as by attachment or fusion to a fluorescent protein.

In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell naturally expresses the target macromolecule and undergoes LLPS. In some embodiments, the cell is induced to express the target macromolecule.

In some embodiments, a cell in the cellular composition expresses the target macromolecule. In some embodiments, expression may include any of gene duplication, transcription, and translation. In some embodiments, the target macromolecule is a polynucleotide, such as an RNA, wherein the target macromolecule is transcribed in a cell in a cellular composition. In some embodiments, the target macromolecule is a polypeptide, such as a protein, wherein the target macromolecule is translated in a cell in a cellular composition. In some embodiments, the target macromolecule is heterologous to the cell.

Many condensates are well known in the art. Examples of known condensates include cleavage bodies, p-granules, histone locus bodies, multivesicular bodies, neuronal RNA granules, nuclear gems, nuclear pores, nuclear speckles, nucleolar caps, nuclear stress bodies, a nucleolus, Octl/PTF/transcription (OPT) domains, paraspeckles, perinucleolar compartments, PML nuclear bodies, PML oncogenic domains, polycomb bodies, processing bodies, Sam68 nuclear bodies, stress granules, or splicing speckles. Numerous condensates are known to form but have not yet been described. Many condensates can be identified using microscopy. In some embodiments, the methods further comprise identifying the one or more phases. In some embodiments, the one or more phases are cellular condensates. In some embodiments, the one or more phases are within one or more cells in the cellular composition. In some embodiments, the one or more phases are one or more stress granules. In some embodiments the one or more phases are one or more nucleolar caps. In some embodiments, the first set of one or more phases are one or more stress granules. In some embodiments, the second set of one or more phases are one or more nuclear paraspeckles, condensates formed around sites of DNA damage, P bodies, Cajal bodies, and PML bodies.

In some embodiments, the condensate is selected from the group consisting of a stress granule, nucleolar caps, P body, Cajal body, PML body, paraspeckle, e.g., a nuclear paraspeckle, DNA damage foci condensate, cleavage body, p-granule, histone locus body, multivesicular body, neuronal RNA granule, nuclear gem, nuclear pore, nuclear stress body, nucleolus, Octl/PTF/transcription (OPT) domain, perinucleolar compartment, PML oncogenic domain, polycomb body, processing body, Sam68 nuclear body, and splicing speckle. Exemplary condensates are discussed in, e.g., Banani et al., Nat Rev Mol Cell Biol, 18, 2017, “Biomolecular condensates: organizers of cellular biochemistry;” Brangwynne et al., Science, 324, 2009, “Germline P granules are liquid droplets that localize by controlled dissolution/condensation;” Patel et al., Cell, 162, 2015, “A Liquid-to-Solid Phase Transition of the ALS Protein Accelerated by Disease Mutation;” Alberti, S., Current Biology, 27, R1089-R1107, 2017, “LLPS in Biology.”

Target macromolecules

In some embodiments, the target macromolecule is a polynucleotide or polypeptide. In some embodiments, the target macromolecule is a polypeptide. In some embodiments, the target macromolecule is a wild-type polypeptide. In some embodiments, the target macromolecule is a mutant polypeptide. In some embodiments, the target macromolecule is FUS, EWSR1 , TIAL1 , PABPC1 , DMPK1 , MBNL1 , or G3BP1 , or a mutant thereof. In some embodiments, the target macromolecule is FUS. In some embodiments, the target macromolecule is DMPK1 . In some embodiments, the target macromolecule is MBNL1 . In some embodiments, the target macromolecule is G3BP1. In some embodiments, the target macromolecule is a protein or nucleic acid. In some embodiments, the target macromolecule is DNA or RNA. In some embodiments, the target macromolecule is mRNA, hnRNA, or non-coding RNA. In some embodiments, the target macromolecule is rRNA, tRNA, IncRNSA, or miRNA. In some embodiments, the target macromolecule is mRNA, hnRNA, non-coding RNA, rRNA, tRNA, IncRNSA, or miRNA. In some embodiments, the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4-ALK, FUS-CHOP, YAP, TAZ or MBNL1 .

Cellular assays

In some embodiments, the cellular composition is a cell culture. In some embodiments, the condensates form in one part of the cell. In some embodiments, the condensates form only in the nucleus. In some embodiments, the condensates form only in the cytoplasm. In some embodiments, the condensates form in a specific type of tissues, e.g., cell type.

In some embodiments, the methods described herein comprise contacting the compound with a cellular composition comprising one or more phases or a cellular composition capable or undergoing LLPS. One of ordinary skill in the art will readily recognize that cellular processes, including the phase of a macromolecule, are dynamic. The methods described herein thus encompass contacting a composition, such as a cellular composition, with a compound at any point in the life cycle of the one or more phases. For examples, the methods encompass contacting a cellular composition with a compound when the target macromolecule is in any location of the cell, in any quantity, or has post-translation modification status, such as the presence, absence, or level of a phosphorylated residue. In some aspects, the methods may also encompass, e.g. contacting a cell with a compounds when the one or more phases are in any location of the cell, are present in any quantity, including being absent, are undergoing a morphological change, such as a change in size of liquidity, or are changing in composition.

In some embodiments, the cellular composition comprises one or more phases prior to contact with the compound. In some embodiments, the method further comprises subjecting the cellular composition to a trigger prior to contacting the compound with the cellular composition comprising one or more phases, or with a cellular composition that is capable or undergoing LLPS. In some embodiments, the cellular composition does not comprise the target macromolecule that have undergone LLPS prior to contact with a compound, and the methods comprises subjecting the cellular composition to a trigger to undergo LLPS after the cellular composition is contacted with the compound. In some embodiments, the cellular composition does not comprise the target macromolecule that has undergone LLPS prior to contact with the compound and undergoes LLPS simultaneously upon contact with the compound. In some embodiments, the LLPS occurs simultaneously and after adding the compound. In some embodiments, the cellular composition is subject to a trigger prior to determining the LLPS of the target macromolecule. In some embodiments, the cellular composition comprises a target macromolecule that has partially undergone LLPS, and additional LLPS occurs simultaneously with contacting the cellular composition with the compound. In some embodiments, the cellular composition comprises a target macromolecule that has undergone LLPS, and additional LLPS occurs simultaneously with contacting the cellular composition with the compound. In some embodiments, the cellular composition comprises a target macromolecule that has partially undergone LLPS, and reverses LLPS simultaneously with contacting the cellular composition with the compound. In some embodiments, the cellular composition comprises a target macromolecule that has undergone LLPS, and reverses LLPS simultaneously with contacting the cellular composition with the compound.

In some embodiments, the methods described herein comprise contacting the cellular composition comprising one or more phases, or a cellular composition capable or undergoing LLPS with a trigger of LLPS. In some embodiments, the microdroplet contains 0.5-6% of the trigger of LLPS. In some embodiments, the trigger of LLPS is PEG1 Ok or a salt, such as NaCI or KCI, among others.

In some embodiments, the conditions to form the condensate comprises the addition of a trigger or the exposure of the cellular composition to a physical stressor. In some embodiments, the method comprises subjecting the cellular composition to any one or more of the following: (a) an oxidative stressor, (b) a mitochondrial electron transport chain inhibitor, (c) a heat stressor, (d) an osmotic stressor, (e) a hyperosmotic stressor (f) glycolysis inhibitor and (g) a salt solution. In some embodiments, the trigger is sodium arsenate, sorbitol, rotenone, 6-deoxyglucose in the absence of glucose, Actinomycin D, or Adenosine dialdehyde (AdOx). In some embodiments, the physical stressor is a heat stressor exposing the cellular composition to a temperature of 40-45°C, such as 43°C. In some embodiments, the physical stressor is an aging condition, e.g., incubation, shaking, and/or heat.

In some embodiments, the method is repeated with different triggers for the same compound, and same cell lines, and the results are compared between the different triggers.

In some embodiments, the reference is a cellular composition that was treated with a reference compound. In some embodiments, the reference is an experimental control. In some embodiments, the reference is a cellular composition that does not contact the compound. In some embodiments, the reference is a cellular composition that was not treated with a trigger or physical stressor. In some embodiments, the reference is prepared in a manner such that a meaningful result can be assessed. For example, in some embodiments, the reference is a cellular composition, wherein the cellular composition is prepared in a similar manner as the cellular composition contacted with the compound, except the reference cellular composition is not subjected to the tested compound or the same step of contacting with the compound. In some embodiments, the reference compound is a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule. In some embodiments, the reference compound is PEGI Ok. In some embodiments, the reference compound is DMSO or absent.

In some embodiments, the reference level is the level of level of modulation of LLPS of the one or more macromolecules in the one or more phases induced by a negative reference compound. In some embodiments, the level of modulation of the LLPS of the compound is equal to the level of modulation of the LLPS of a negative reference compound. In some embodiments, the level of modulation of the LLPS of the compound is higher than the level of modulation of the LLPS of a negative reference compound. In some embodiments, the negative reference compound is a compound that does not induce LLPS of one or more macromolecules in the one or more phases. In some embodiments, the negative reference compound is DMSO.

In some embodiments, the level of modulation of LLPS of the one or more macromolecules in the one or more phases is determined based off any one or more of the following: (i) the number of target molecules that have undergone LLPS; (ii) size of the one or more phases; (iii) location of the one or more phases; (iv) distribution of one or more phases; (v) surface area of the one or more phases; (vi) composition of the one or more phases, (vii) liquidity of the one or more phases; (viii) solidification of the one or more phases; (ix) dissolution of the one or more phases; (x) location of the target macromolecule; (xi) partitioning of the target macromolecule; and (xii) aggregation of the target macromolecule.

In some embodiments, the determining is performed within about 60 days of the contacting of the compound with the cellular composition, such as within about 35 days, about 28 days, about 21 days, about 14 days, about 10 days, about 7 days, about 5 days, about 3 days, about 2 days, about 1 day, about 12 hours about 5 hours, about 2 hours, about 1 hour, about 45 minutes, about 30 minutes, about 15 minutes, about 5 minutes, about 1 minute, or about 30 seconds. In some embodiments, the determining is performed after about 5 seconds of the contacting of the compound with the cellular composition, such as, after about 15 seconds, about 30 seconds, about 1 minute, about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 5 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 5 days, about 7 days, about 10 days, about 14 days, about 21 days, about 28 days, about 35 days, or about 60 days.

In some embodiments, the method further comprises repeating the determining step of the method. For example, in some embodiments, the methods comprise repeating the determining step of the method at least 2, 3, 4, 5, 10, or more times. In some embodiments, the method comprises performing the determining step for a first portion of the cellular composition and a second portion of the cellular composition, such as a first cell and a second cell in the cellular composition. In some embodiments, the method comprises performing the determining step for a third, fourth, fifth, sixth, or more portion of the cellular composition, such as a third, fourth, fifth, sixth, or more cell in the cellular composition. In some embodiments the method comprises performing the determining step for a first portion of a cell in the cellular composition and a second portion of a cell in the cellular composition, such as in the cytoplasm and the nucleus, or in a first organelle and a second organelle. In some embodiments, the method comprises performing the determining step for a third, fourth, fifth, sixth, or more portion of a cell in the cellular composition.

In some embodiments, the determining step of the method is repeated after an interval of time, such as about 30 seconds, about 1 minute, about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 5 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 5 days, about 7 days, about 10 days, about 14 days, about 21 days, about 28 days, about 35 days, about 60 days, or more. In some embodiments the determining step is based on the same characteristic when repeated. In some embodiments, the method further comprises comparing the characteristic over time, such as comparing the number of condensates with one day between determinations.

In some embodiments, the method is repeated with various concentrations of the compound. In some embodiments, the results from different concentrations of the same compound are compared. In some embodiments, the method is repeated with various concentrations of the one or more macromolecules. In In some embodiments, the results from different concentrations of the same macromolecule are compared.

In some embodiments, the cellular compositions, and condensates are visualized by microcopy, including for example, stereoscopic microscopy, brightfield microscopy, polarizing microscopy, phase contrast microscopy, differential interference contrast microscopy, fluorescence microscopy, total internal reflection fluorescence microscopy, confocal microscopy, or multiphoton excitation microscopy. In some embodiment, the visualization is performed continuously on the stream of microdroplets. In some embodiments, the microdroplets and condensates are visualized by multi-color epifluorescence microscope. In some embodiments the multi-color epifluorescence microscope excites the microdroplets and detects a response. In some embodiments, the multi-color epifluorescence microscope excites and detects a response on multiple different fluorescence channels. In some embodiments, the multiple fluorescence channels are 470nm, 555nm and 640 nm. In some embodiments, the excitation is light via a laser diode. In some embodiments, the laser diode emits a wavelength of 470nm, 555nm, or 640 nm. In some embodiments, the relative concentrations of the compounds and each of the target macromolecules are determined based on the respective responses of each target macromolecules to the excitation. In some embodiments, the relative concentration of the compounds is determined from the fluorescent intensity of Alexa 546 or Alexa 647 dyes. In some embodiments, the relative concentration of each of the target macromolecules comprise a different fluorophore which emits a specific wavelength in response to excitation.

In some embodiments, the multi-color epifluorescence microscope obtains a light-scattering profile of the cellular composition. In some embodiments, the light-scattering profile of the cellular composition determines the particular phases of the target macromolecules. In some embodiments, the relative concentrations of each of the target macromolecules of the cellular composition are varied based on the measured relative concentrations of the target macromolecule, and the phases of the macromolecule present in the cellular composition s. In some embodiments, the relative concentrations of the constituents of the cellular composition are systematically varied.

In some embodiments, the condensate and/or target macromolecules may be labeled, for example, labeled with a fluorophore. In some embodiments, the target macromolecule comprises a fluorescent dye, such as a fluorescent protein, e.g., GFP, RYP, or YFP. In some embodiments, the method comprises contacting at least a portion of the cellular composition with a label. In some embodiments, the label is a labeled binding molecule, such as an antibody or biotin-binding protein. In some embodiments, the label is a stain, such as a stain specific to an organelle.

In some embodiments, the method further comprises imaging at least a portion of the cellular composition, such as a field of view. In some embodiments, the method further comprises contacting at least a portion of the cellular composition with a fixative. In some embodiments, the method further comprises contacting at least a portion of the cellular composition with a stain. In some embodiments, the method further comprises contacting at least a portion of the cellular composition with a DNA- damaging condition. In some embodiments, the DNA-damaging condition is laser irradiation.

The LLPS of target macromolecules can be determined for a portion or all of the cellular composition. Accordingly, in some embodiments, the method comprises determining the LLPS of the target macromolecule in a portion of the cellular composition. In some embodiments, the method comprises determining the LLPS of the target macromolecule in the entire cellular composition. In some embodiments, the method comprises determining the LLPS of the target macromolecule in one or more cells in the cellular composition. In some embodiments, the method comprises determining the LLPS of the target macromolecule in a single cell in the cellular composition.

The LLPS of target macromolecules can also be determined for a portion or all of a cell in the cellular composition. Accordingly, in some embodiments, the method comprises determining the LLPS of the target macromolecule in a portion of one or more cells in the cellular composition. In some embodiments, the method comprises determining the LLPS of the target macromolecule in a portion of a single cell in the cellular composition. In some embodiments, the method comprises determining the LLPS of the target macromolecule in the cytoplasm. In some embodiments, the method comprises determining the LLPS of the target macromolecule in the nucleus. In some embodiments, the method comprises determining the LLPS of the target macromolecule in an organelle.

In some embodiments, the LLPS of the target macromolecule increases as compared to the reference. In some embodiments, the LLPS of the target macromolecule decreases as compared to the reference. In some embodiments, the LLPS of the target macromolecule in the cytoplasm decreases compared to the reference and the LLPS of the target macromolecule in the nucleus does not decrease compared to the reference. In some embodiments, the LLPS of the target macromolecule in the cytoplasm decreases compared to the reference and the LLPS of the target macromolecule in the nucleus increases compared to the reference.

In some embodiments, the compound is suramin, the one or more macromolecules is FUS, the triggers are sodium arsenite (NaArse), Actinomycin D, or Adenosine dialdehyde (AdOx), and the condensate is nucleolar caps.

Examples

Exemplary embodiments have been disclosed above and illustrated in the accompanying drawings. It will be understood by those skilled in the art that various changes, omissions, and additions may be made to that which is specifically disclosed herein without departing from the spirit and scope of the present invention.

Example 1. Phase Boundary Shifts of Compounds

Compound A is injected onto a microfluidic chip at various concentrations with various target macromolecules (such as DDX3X, G3BP1 , YAP, YTHDC1 or EML4-ALK) at various concentrations. The microfluidic chip is assayed, and a phase boundary shift is measured for each protein as shown in FIG. 2.

Results: a compound is identified against the EML4-ALK condensate as selective over other condensate systems (DDX3X, G3BP1 , YAP, YTHDC1 ).

Example 2: Molecular screening assay for different binding modes

Three compounds (Compound B, Compound C, Compound D) were injected onto a microfluidic chip comprising G3BP1 -RNA condensates. All three were found to modulate the phase transition as shown in FIG. 3A. The compound-condensate systems were further assayed by a microscale thermophoresis assay as shown in FIG. 3B and by microfluidic diffusional sizing as shown in FIG. 3C.

Results: Compound C binds the protein directly and Compound B disrupts the protein-RNA interaction.

Example 3. Suramin screening

Multiple compositions of FUS-GFP, different concentrations of suramin between 1 .2 pM and 25 pM, were pre-mixed into solution prior to injection onto a microfluidic chip. The relative concentration of the FUS condensates and the compound was measured . Negative controls of 1% v/v DMSO were included, and the resultant phase boundary was compared to the negative control was shown in FIG. 4A. Example 4. Cellular Assay

FUS-GFP Hela cell line were grown in culture. These cells were exposed to either DMSO control or a trigger. The trigger was 0.1 pM Actinomycin D. Suramin was exposed individually at concentrations of 5 pM and 20 pM to each of the cell cultures for a predetermined amount of time. For some compounds, this procedure was repeated at more than one concentration. The cells were observed for GFP fluorescence as shown in FIG. 4C. Optionally, Alexa 546 and Alexa 647 dyes were used to visualize FUS, or G3BP1 protein.

Results: Suramin leads to condensate dissolution in cells.

Example 5. Suramin screening with functional effects

HeLa cells were treated as in Example 4. Then, the cells underwent RNAseq to identify downregulation or upregulation of genetic pathways. Treatment of those cells with Suramin, followed by further RNAseq to identify downregulation or upregulation of genetic pathways as shown in FIG. 5.

Results: Suramin leads to upregulation of these genes (RNA processing, the nucleolus, gene silencing and negative regulation of gene expression) that contributes towards the restoration of the desired phenotype.

Example 6. Suramin screening of cells with FUS Mutations

Cells with FUS mutations (P525L and R495X) were treated with 500 pM Suramin after the cells were treated with 0.1 pM Actinomycin D as shown in FIG. 6.

Results: These cell lines were resistant to Suramin treatment.

Example 7. Microfluidic droplet generation and imaging

Compositions comprising a compound, one or more macromolecules, and buffer are generated in the well plate autosampler as shown in FIG. 7 and the composition, in varying relative concentrations, is injected onto the microfluidic chip, by flow rates controlled by a flow control system and co-encapsulated in microdroplets generated by a flow-focusing microfluidic device. The microdroplets are incubated for a predetermined amount of time during passage through an incubation flow chamber, before undergoing imaging by a three-laser diode and beam splitter to enable the simultaneous excitation and acquisition of green, red, and far-red fluorescence. The phase separation is probed inside the microdroplets to create a phase separation boundary to identify modulators of interest.

Example 8. Compound library screening

FUS-GFP, 0.5-6% w/v PEG10k, one of 1029 compounds from an FDA-approved repurposing library in a concentration between about 1 .2 pM and 100 pM, and Alexa 546, or Alexa 647 dyes were injected onto a microfluidic chip as described in Example 7. The relative concentration of the FUS-GFP was determined by fluorescence labeling strategy and quantitative multi-color fluorescence imaging as shown in FIG. 7. The PEG10k acts as a trigger of LLPS and the FUS-GFP fluorescence determined the phase separated or homogenous phases of the protein by a droplet classification algorithm as shown in FIG. 7. Within each screening set, negative reference microdroplet comprising 1 % v/v DMSO, and the positive reference microdroplet comprising 5% w/v 1 ,6 hexanediol, a trigger of FUS, were included.

Example 9. In silico screening

Using the compositions and methods described herein, one can utilize in silico screening procedures to identify modulators of biomolecular targets, as well as therapeutically relevant biomolecular targets that are amenable to phase transition modulation. For example, FIG. 8 is a schematic diagram of an exemplary silico screening process described herein. The biomolecular condensate target screened in the molecular I in vitro assay (FIG. 1 , box #6) may be identified via an in-silico screening process that brings omics data from human samples and cellular models together with biomolecular condensate related data, such as data acquired via imaging, spatial transcriptomics I proteomics, and biochemical assays, among others. The latter data can be used to develop models that predict the condensation behavior of targets which do not have their condensation behavior profiled, their mutated forms or under conditions (e.g. cell lines, stressed conditions, etc.) for which experimental data is unavailable

FIG. 1 1 shows an example of how the strategy outlined in FIGS. 9-10 is reduced to practice to identify condensate targets across a variety of patient cohorts. Genetic data across the coding genome is analyzed for the cohort of interest to identify cases where mutations are enriched into the regions that are important for the phase separation process. The process identifies CTNNB1 as a condensate target of interest as mutations are enriched into regions important for the phase separation process. Optionally, it may be required that these mutations are not present or are present much less frequently among health volunteers.

Enumerated Embodiments

The invention set forth in the present disclosure is also represented by the following exemplary, non-limiting, enumerated embodiments:

E1 . A method of identifying condensate modulators, the method comprising contacting a condensate modulator with a composition comprising a target macromolecule and measuring one or more phase transition characteristics of the condensate.

E2. The method of embodiment 1 , wherein the method comprising:

(a) producing a stream of microdroplets, wherein each of said microdroplets comprises a compound and the target macromolecule, wherein the concentration of the compound varies among the plurality of microdroplets;

(b) determining, within each of the microdroplets, one or more phase transition characteristics of the condensate; and

(c) determining whether the compound modulates one or more phase transition characteristics of the condensate, wherein: the determining is performed by comparing the concentration of the compound at which the one or more phase transition characteristics change the one or more phase transition characteristics change as compared to a concentration of a reference compound at which the one or more phase transition characteristics change the one or more phase transition characteristics change.

E3. The method of embodiment 1 or 2, wherein the microdroplet comprises a trigger of phase transition of a condensate that is known to modulate one or more phase transition characteristics of a condensate in vitro.

E4. The method of any one of embodiments 1 -3, wherein the reference compound is a trigger of phase transition of a condensate that is known to modulate one or more phase transition characteristics of a condensate. E5. The method of embodiment 2 or 3, wherein the trigger of phase transition is a protein, nucleic acid, salt, polyethylene glycol (PEG) or a biological mixture, optionally wherein the biological mixture is a cell lysate.

E6. The method of embodiment 5, wherein the polyethylene glycol (PEG) has an average molecular weight of from 600 Da to 20 kDa or about 10 kDa.

E7. The method of any one of embodiments 1 -6, wherein the target macromolecule is a protein or nucleic acid.

E8. The method of embodiment 7, wherein the nucleic acid is DNA or RNA.

E9. The method of embodiment 8, wherein the RNA is mRNA, hnRNA, non-coding RNA, rRNA, tRNA, IncRNSA, or miRNA.

E10. The method of any one of embodiments 1 -9, wherein the method comprises the step of producing the microdroplets on a microfluidic chip.

E11 . The method of any one of embodiments 1 -10, wherein each microdroplet comprises a portion of a stock mixture comprising the target macromolecule and a portion of a stock mixture comprising the compound.

E12. The method of embodiment 11 , wherein each microdroplet further comprises a portion of a stock mixture comprising the trigger.

E13. The method of any one of embodiments 10-12, wherein each microdroplet is produced by automated mixing of the stock mixtures.

E14. The method of any one of embodiments 11 -13, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration; and each of the target macromolecule is, independently, covalently, or noncovalently bound to a fluorescent dye (FDtarget), whereby fluorescence of one fluorescent dye (FDtarget) facilitates detection of one target macromolecule.

E15. The method of any one of embodiment 11 -14, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the concentration of the target macromolecule in the microdroplet; each of the target macromolecule is, independently, covalently, or noncovalently bound to a fluorescent dye (FDtarget), whereby fluorescence of each fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and the stock mixture comprising the trigger further comprises a fluorescent dye (FDtrigger) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDtrigger) correlates with the concentration of the trigger and facilitates detection of the trigger concentration.

E16. The method of embodiment 14 or 15, wherein each fluorescent dye is different from one another.

E17. The method of embodiment 16, wherein each fluorescent dye exhibits distinct excitation and emission spectra from one another.

E18. The method of any one of embodiments 14-17, wherein each fluorescent dye is selected from 7- nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUETM dye), 5-(dimethylamino)naphthalene-1 -sulfonyl (Dansyl), pyrene, 7-amino-3-{[(2,5- dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350TM), 6,8-difluoro-7-hydroxy-4-methylcoumarin (MARINA BLUETM dye), N-(2-aminoethyl)-4-{5-[4- (dimethylamino)phenyl]-1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYLTM dye), 2, 3,5,6- Tetramethyl-1 H,7H-pyrazolo[1 ,2-a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4- (ethylamino)naphthalen-2-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUETM dye), tris(N,N-diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrolidin-1 -yl)oxy]carbonyl}piperidin- 1 -yl)-2-oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405TM), N,N-diethylethanaminium [9-{6- [(2,5-dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H- benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430TM), 1 -[({4-[(7-nitro-2,1 ,3- benzoxadiazol-4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSYTM dye), fluorescein, 2-(6- amino-3-iminio-4,5-disulfonato-3H-xanthen-9-yl)-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488TM), 2',7'-Difluoro-3',6'-dihydroxy-3H-spiro[isobenzofuran-1 ,9'-xanthen]-3-one (OREGON GREENTM 488), 1 ,3,5,7, 8-pentamethyl-4,4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPYTM 493/503), rhodamine green, 13-[2-carboxy-3,4,6-trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 - yl)oxy-6-oxohexyl]amino]-2-oxoethyl]sulfanylphenyl]-7,7,9,17,19,19-hexamethyl-2-oxa-6,20- diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa-1 (14),3,5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546TM), 2-[5-[3,3-dimethyl-5-sulfo-1 -(3-sulfopropyl)indol-1 -ium-2-yl]penta-2,4- dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5- sulfonic acid (ALEXA FLUOR 647TM), and rhodamine red; and/or wherein one or more of the fluorescent dyes is a fluorescent protein selected from GFP, mNeonGreen, mRFP, mCherry, and mPlum. E19. The method of any one of embodiments 1 -18, wherein the step of determining, within each of the microdroplets, the target macromolecule is visualized by fluorescent microscopy to determine one or more phase transition characteristics of the condensate.

E20. The method of embodiment 19, wherein a finding that the target macromolecule is present within the microdroplet in discrete puncta is taken as an indication that one or more phase transition characteristics of the condensate have changed, and a finding that the target macromolecule is present in a homogenous distribution within the microdroplet, absent discrete puncta, is taken as an indication that one or more phase transition characteristics of the condensate have not changed.

E21 . The method of any one of embodiments 2-20, wherein steps (a) through (c) are performed on a plurality of compounds, thereby screening a compound library to identify a compound that modulates one or more phase transition characteristics of the condensate in vitro.

E22. The method of embodiment 21 , wherein the screening is high-throughput screening.

E23. The method of embodiment 21 or 22, wherein the compound library comprises from 10 to 100,000 compounds, or more.

E24. The method of any one of embodiments 1 -23, wherein the one or more phase transition characteristics are determined from a series of phase diagrams of the condensate.

E25. The method of any one of embodiments 2-24, wherein the concentration of the compound at which the condensate undergoes phase transition is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet, wherein the positive reference microdroplet comprises the target macromolecule and a trigger of phase transition, and the negative reference microdroplet comprises the target macromolecule.

E26. The method of any one of embodiments 1 -25, wherein the compound is identified as one that binds to and/or modulates the activity of the target macromolecule.

E27. The method of any one of embodiments 1 -26, wherein the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4-ALK, FUS-CHOP, YAP, TAZ or MBNL1 .

E28. The method of any one of embodiments 1 -27, wherein the microdroplet comprises a cell lysate, or cells.

E29. The method of any one of embodiments 1 -28, wherein the cells are from a diseased cell line. E30. The method of any one of embodiments 1 -29, wherein the microdroplet comprises a non-target macromolecule; optionally wherein the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that modulates phase transition characteristics of a condensate system that comprises the target macromolecule.

E31 . The method of embodiment 30, wherein the non-target macromolecule is an RNA transcript.

E32. The method of embodiment 31 , wherein the non-target macromolecule is a specific RNA, a DMPK transcript or AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

E33. The method of embodiment 1 , the method further comprising:

(a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, and wherein the concentration of the compound varies among the plurality of microdroplets; .

(c) determining whether the compound modulates one or more phase transition characteristics of the condensate, wherein: the determining is performed by comparing the concentration of the compound at which the one or more phase transition characteristics change as compared to a concentration of a reference compound at which the one or more phase transition characteristics change;

(a’) introducing, into a cell, the compound identified in (c) as one that modulates one or more phase transition characteristics of the condensate in vitro, wherein the cell comprises the target macromolecule; and

(b’) assessing whether the compound modulates one or more phase transition characteristics of the condensate in the cell, wherein: the determining is performed by comparing the concentration of the compound at which the one or more phase transition characteristics change in the cell, as compared to a concentration of a reference compound at which the one or more phase transition characteristics change in the cell; optionally wherein the one or more phase transition characteristics correlates with a change in the functional activity of the condensate.

E34. The method of embodiment 33, wherein the microdroplet comprises a trigger of phase transition of a condensate that is known to modulate one or more phase transition characteristics of a condensate in vitro.

E35. The method of embodiment 34, wherein a trigger of phase transition that is known to modulate one or more phase transition characteristics of a condensate in a cell, is added to the cell. E36. The method of any one of embodiments 33-35, wherein the reference compound is a trigger of phase transition of a condensate that is known to modulate one or more phase transition characteristics of a condensate.

E37. The method of any one of embodiments 34-36, wherein the trigger of phase transition is a protein, nucleic acid, salt, polyethylene glycol (PEG) or a biological mixture, optionally wherein the biological mixture is a cell lysate.

E38. The method of embodiment 37, wherein the polyethylene glycol (PEG) has an average molecular weight of from 600 Da to 20 kDa or about 10 kDa.

E39. The method of any one of embodiments 33-38, wherein the target macromolecule is a protein or nucleic acid.

E40. The method of embodiment 39, wherein the nucleic acid is DNA or RNA.

E41 . The method of embodiment 40, wherein the RNA is mRNA, hnRNA, non-coding RNA, rRNA, tRNA, IncRNSA, or miRNA.

E42. The method of any one of embodiments 33-41 , wherein the method comprises the step of producing the microdroplets on a microfluidic chip.

E43. The method of any one of embodiments 33-42, wherein each microdroplet comprises a portion of a stock mixture comprising the target macromolecule and a portion of a stock mixture comprising the compound.

E44. The method of embodiment 43, wherein each microdroplet further comprises a portion of a stock mixture comprising the trigger.

E45. The method of any one of embodiments 42-44, wherein each microdroplet is produced by automated mixing of the stock mixtures.

E46. The method of any one of embodiments 33-45, wherein the compound is added to the cell as a stock mixture comprising the compound.

E47. The method of embodiment 46, wherein the trigger is added to the cell as a portion of a stock mixture comprising the trigger.

E48. The method of any one of embodiments 43-47, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration; and each of the target macromolecule is, independently, covalently, or noncovalently bound to a fluorescent dye (FDtarget), whereby fluorescence of one fluorescent dye (FDtarget) facilitates detection of one target macromolecule.

E49. The method of any one of embodiment 43-48, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the concentration of the target macromolecule in the microdroplet; each of the target macromolecule is, independently, covalently, or noncovalently bound to a fluorescent dye (FDtarget), whereby fluorescence of each fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and the stock mixture comprising the trigger further comprises a fluorescent dye (FDtrigger) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDtrigger) correlates with the concentration of the trigger and facilitates detection of the trigger concentration.

E50. The method of embodiment 48 or 49, wherein each fluorescent dye is different from one another.

E51 . The method of embodiment 50, wherein each fluorescent dye exhibits distinct excitation and emission spectra from one another.

E52. The method of any one of embodiments 48-51 , wherein each fluorescent dye is selected from 7- nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUETM dye), 5-(dimethylamino)naphthalene-1 -sulfonyl (Dansyl), pyrene, 7-amino-3-{[(2,5- dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350TM), 6,8-difluoro-7-hydroxy-4-methylcoumarin (MARINA BLUETM dye), N-(2-aminoethyl)-4-{5-[4- (dimethylamino)phenyl]-1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYLTM dye), 2, 3,5,6- Tetramethyl-1 H,7H-pyrazolo[1 ,2-a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4- (ethylamino)naphthalen-2-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUETM dye), tris(N,N-diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrolidin-1 -yl)oxy]carbonyl}piperidin- 1 -yl)-2-oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405TM), N,N-diethylethanaminium [9-{6- [(2,5-dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H- benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430TM), 1 -[({4-[(7-nitro-2,1 ,3- benzoxadiazol-4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSYTM dye), fluorescein, 2-(6- amino-3-iminio-4,5-disulfonato-3H-xanthen-9-yl)-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488TM), 2',7'-Difluoro-3',6'-dihydroxy-3H-spiro[isobenzofuran-1 ,9'-xanthen]-3-one (OREGON GREENTM 488), 1 ,3,5,7, 8-pentamethyl-4,4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPYTM 493/503), rhodamine green, 13-[2-carboxy-3,4,6-trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 - yl)oxy-6-oxohexyl]amino]-2-oxoethyl]sulfanylphenyl]-7,7,9,17,19,19-hexamethyl-2-oxa-6,20- diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa-1 (14),3,5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546TM), 2-[5-[3,3-dimethyl-5-sulfo-1 -(3-sulfopropyl)indol-1 -ium-2-yl]penta-2,4- dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5- sulfonic acid (ALEXA FLUOR 647TM), and rhodamine red;

And/or wherein one or more of the fluorescent dye is a fluorescent protein selected from GFP, mNeonGreen, mRFP, mCherry, and mPlum.

E53. The method of any one of embodiments 33-52, wherein the step of determining, within each of the microdroplets, the target macromolecule is visualized by fluorescent microscopy to determine one or more phase transition characteristics of the condensate.

E54. The method of embodiment 53, wherein a finding that the target macromolecule is present within the microdroplet in discrete puncta is taken as an indication that one or more phase transition characteristics of the condensate have changed, and a finding that the target macromolecule is present in a homogenous distribution within the microdroplet, absent discrete puncta, is taken as an indication that one or more phase transition characteristics of the condensate have not changed.

E55. The method of any one of embodiments 33-54, wherein steps (a) through (c) are performed on a plurality of compounds, thereby screening a compound library to identify a compound that modulates one or more phase transition characteristics of the condensate in vitro.

E56. The method of any one of embodiments 33-55, wherein steps (a’) through (b’) have been performed on a plurality of compounds, thereby screening a compound library to identify a compound that modulates the one or more phase transition characteristics of the condensate in the cell.

E57. The method of embodiment 56, wherein the screening is high-throughput screening.

E58. The method of any one of embodiments 55-57, wherein the compound library comprises from 10 to 100,000 compounds, or more.

E59. The method of any one of embodiments 33-58, wherein the one or more phase transition characteristics are determined from a series of phase diagrams of the condensate.

E60. The method of any one of embodiments 33-59, wherein the concentration of the compound at which the condensate undergoes phase transition is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet, wherein the positive reference microdroplet comprises the target macromolecule and a trigger of phase transition, and the negative reference microdroplet comprises the target macromolecule.

E61 . The method of any one of embodiments 33-60, wherein the compound is identified as one that binds to and/or modulates the activity of the target macromolecule.

E62. The method of any one of embodiments 33-61 , wherein the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4-ALK, FUS-CHOP, YAP, TAZ or MBNL1 .

E63. The method of any one of embodiments 33-62, wherein the microdroplet comprises a cell lysate or cells.

E64. The method of embodiment 63, wherein the cells are from a diseased cell line.

E65. The method of any one of embodiments 33-64, wherein the microdroplet comprises a non-target macromolecule; optionally wherein the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that modulates phase transition characteristics of a condensate system that comprises the target macromolecule.

E66. The method of embodiment 65, wherein the non-target macromolecule is an RNA transcript.

E67. The method of embodiment 66, wherein the non-target macromolecule is a specific RNA, a DMPK transcript or AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

E68. The method of any one of embodiments 33-67, wherein the concentration of the compound at which the one or more phase transition characteristics change is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet, wherein the positive reference microdroplet comprises the target macromolecule and a trigger of phase transition, and the negative reference microdroplet comprises the target macromolecule.

E69. The method of any one of embodiments 33-68, wherein the cell is a eukaryotic cell.

E70. The method of embodiment 69, wherein the eukaryotic cell is a mammalian cell.

E71 . The method of embodiment 70, wherein the mammalian cell is a human cell.

E72. The method of any one of embodiments 33-71 , wherein the cell naturally expresses the target macromolecule and undergoes phase transition. E73. The method of any one of embodiments 33-72, wherein the cell is induced to express the target macromolecule.

E74. The method of any one of embodiments 34-73, wherein the trigger induces oxidative stress in the cell, thereby promoting localization of the target macromolecule to cytoplasmic stress granules.

E75. The method of embodiment 74, wherein the trigger induces oxidative stress is sodium arsenite.

E76. The method of any one of embodiments 34-75, wherein the trigger induces a change in a chemical modification state of the target macromolecule, thereby causing the target macromolecule to localize to a condensate in the cell.

E77. The method of embodiment 76, wherein the chemical modification is a post-transcriptional modification, such as phosphorylation, methylation, or acetylation.

E78. The method of embodiment 77, wherein the chemical modification is methylation, and wherein the trigger induces a change in methylation state is adenosine dialdehyde.

E79. The method of any one of embodiments 34-78, wherein the trigger induces formation of nucleolar caps in the cell, thereby promoting localization of the target macromolecule to a nucleolar cap.

E80. The method of embodiment 79, wherein the trigger induces formation of nucleolar caps in the cell is actinomycin D.

E81 . The method of any one of embodiments 33-80 wherein step (b’) is performed by way of fluorescent microscopy.

E82. The method of any one of embodiments 33-81 , wherein the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4-ALK, FUS-CHOP, YAP, TAZ or MBNL1 .

E83. A method of identifying selective condensate modulators, the method comprising contacting a condensate modulator with a composition comprising a target macromolecule and a non-target macromolecule, and measuring one or more phase transition characteristics of the condensate.

E84. The method of embodiment 83, the method comprising:

(a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, a non-target macromolecule and wherein the concentration of the compound varies among the plurality of microdroplets; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (Donn-target), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule;

(b) determining, within each of the microdroplets, whether the target macromolecule has undergone phase transition, and whether the non-target macromolecule has undergone phase transition, thereby identifying the concentration of the compound at which the one or more phase transition characteristics change and the concentration of the compound at which the non-target macromolecule undergoes phase transition;

(c) determining whether the compound selectively modulates one or more phase transition characteristics of the condensate, wherein: the determining is by comparing the concentration of the compound at which the one or more phase transition characteristics change as compared to a concentration of at which the non- target macromolecule undergoes phase transition.

E85. The method of embodiment 84, wherein the microdroplet comprises a trigger of phase transition of a condensate that is known to modulate one or more phase transition characteristics of a condensate in vitro.

E86. The method of embodiment 84 or 85, wherein the reference compound is a trigger of phase transition of a condensate that is known to modulate one or more phase transition characteristics of a condensate.

E87. The method of embodiment 85 or 86, wherein the trigger of phase transition is a protein, nucleic acid, salt, polyethylene glycol (PEG) or a biological mixture, optionally wherein the biological mixture is a cell lysate.

E88. The method of any one of embodiments 84-87, wherein the target macromolecule is a protein or nucleic acid.

E89. The method of embodiment 88, wherein the nucleic acid is DNA or RNA.

E90. The method of embodiment 89, wherein the RNA is mRNA, hnRNA, non-coding RNA, rRNA, tRNA, IncRNSA, or miRNA.

E91 . The method of any one of embodiments 84-90, wherein the non-target macromolecule is a protein or a nucleic acid. E92. The method of embodiment 91 , wherein the non-target macromolecule is an RNA transcript, a specific RNA, a DMPK transcript, an AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

E93. The method of any one of embodiments 84-92, wherein the method comprises the step of producing the microdroplets on a microfluidic chip.

E94. The method of any one of embodiments 84-93, wherein each microdroplet comprises a portion of a stock mixture comprising the target macromolecule, a portion of a stock mixture comprising the compound and a position of a stock mixture comprising a non-target macromolecule.

E95. The method of embodiment 94, wherein each microdroplet further comprises a portion of a stock mixture comprising the trigger.

E96. The method of embodiment 94 or 95, wherein each microdroplet is produced by automated mixing of the stock mixtures.

E97. The method of any one of embodiments 94-96, wherein:

The stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration.

E98. The method of any one of embodiments 95-97, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration; and the stock mixture comprising the trigger further comprises a fluorescent dye (FDtrigger) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDtrigger) correlates with the concentration of the trigger and facilitates detection of the trigger concentration.

E99. The method of embodiment 97 or 98, wherein each fluorescent dye is different from one another.

E100. The method of embodiment 99, wherein each fluorescent dye exhibits distinct excitation and emission spectra from one another.

E101 . The method of any one of embodiments 97-100, wherein each fluorescent dye is selected from 7-nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUETM dye), 5-(dimethylamino)naphthalene-1 -sulfonyl (Dansyl), pyrene, 7-amino-3-{[(2,5- dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350TM), 6,8-difluoro-7-hydroxy-4-methylcoumarin (MARINA BLUETM dye), N-(2-aminoethyl)-4-{5-[4- (dimethylamino)phenyl]-1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYLTM dye), 2, 3,5,6- Tetramethyl-1 H,7H-pyrazolo[1 ,2-a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4- (ethylamino)naphthalen-2-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUETM dye), tris(N,N-diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrolidin-1 - yl)oxy]carbonyl}piperidin-1 -yl)-2-oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405TM), N,N- diethylethanaminium [9-{6-[(2,5-dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4- (trifluoromethyl)-8,9-dihydro-2H-benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430TM), 1 - [({4-[(7-nitro-2,1 ,3-benzoxadiazol-4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSYTM dye), fluorescein, 2-(6-amino-3-iminio-4, 5-disulfonato-3H-xanthen-9-yl)-5-((2, 3,5,6- tetrafluorophenoxy)carbonyl)benzoate (ALEXA ’LU’R 488TM), 2’,7’-Difluoro-3',6'-dihydroxy-3H- spiro[’sobenzofuran-1 ,9'-xanthen]-3-one (OREGON GREENTM 488), 1 ,3,5,7, 8-pentamethyl-

4.4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPYTM 493/503), rhodamine green, 13-[2- carboxy-3,4,6-trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 -yl)oxy-6-oxohexyl]amino]-2- oxoethyl]sulfanylphenyl]-7,7,9,17,19,19-hexamethyl-2-oxa-6,20- diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa-1 (14),3,5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546TM), 2-[5-[3,3-dimethyl-5-sulfo-1 -(3-sulfopropyl)indol-1 -ium-2-yl]penta-

2.4-dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5- sulfonic acid (ALEXA FLUOR 647TM), and rhodamine red;

E102. The method of any one of embodiments 84-101 , wherein the step of determining, within each of the microdroplets, the target macromolecule is visualized by fluorescent microscopy to determine the phase transition characteristics of the condensate.

E103. The method of any one of embodiments 84-102, wherein method identifies the compound that modulates the phase characteristics of the condensate.

E104. The method of any one of embodiments 84-103, wherein the method identified the compound that selectively modulates heterotypic phase transition of the target macromolecule and the non-target macromolecule.

E105. The method of embodiment 104, wherein a finding that the target macromolecule is present within the microdroplet in discrete puncta is taken as an indication that the phase transition characteristics of the condensate have changed, and a finding that the target macromolecule is present in a homogenous distribution within the microdroplet, absent discrete puncta, is taken as an indication that the phase transition characteristics of the condensate have not changed. E106. The method of any one of embodiments 84-105, wherein steps (a) through (c) are performed on a plurality of compounds, thereby screening a compound library to identify a compound that modulates the phase transition characteristics of the condensate in vitro.

E107. The method of any one of embodiments 84-106, wherein the screening is high-throughput screening.

E108. The method of any one of embodiments 106-107, wherein the compound library comprises from 10 to 100,000 compounds, or more.

E109. The method of any one of embodiments 84-108, wherein the concentration of the compound at which the one or more phase transition characteristics change is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet,

Wherein the positive reference microdroplet comprises the target macromolecule and a trigger of phase transition, and the negative reference microdroplet comprises the target macromolecule.

E110. The method of any one of embodiments 84-109, wherein the microdroplet comprises a cell lysate or cells.

E111 . The method of embodiment 110, wherein the cells are from a diseased cell line.

E112. The method of any one of embodiments 84-111 , wherein the microdroplet comprises a non-target macromolecule.

E113. The method of embodiment 112, wherein the non-target macromolecule is an RNA transcript.

E114. The method of embodiment 113, wherein the non-target macromolecule is a specific RNA, a DMPK transcript or AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

E115. The method of any one of embodiments 84-114, wherein the compound is identified as one that binds to, and/or modulates the activity of the target macromolecule.

E116. The method of any one of embodiments 112-114, wherein:

(a) the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that modulates heterotypic phase separation of the target macromolecule and the non-target macromolecule; or

(b) the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that modulates phase transition characteristics of a condensate system that comprises the target macromolecule.

E117. The method of any one of embodiments 84-116, wherein the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4-ALK, FUS-CHOP, YAP, TAZ or MBNL1 .

E118. A method of identifying selective condensate modulators, the method comprising contacting a condensate modulator with a composition comprising a target macromolecule and a non-target macromolecule, and measuring one or more phase transition characteristics of the condensate.

E119. The method of embodiment 118, the method comprising:

(a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, a non-target macromolecule and wherein the concentration of the compound varies among the plurality of microdroplets; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-target), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule,

(c) determining whether the compound selectively modulates one or more phase transition characteristics of the condensate, wherein: the determining is by comparing the concentration of the compound at which the one or more phase transition characteristics change as compared to a concentration of at which the non- target macromolecule undergoes phase transition,

(a’) introducing, into a cell, the compound identified in (c) as one that selectively modulates one or more phase transition characteristics of the condensate, in vitro, wherein the cell comprises the target macromolecule and the non-target macromolecule; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-target), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, and

(b’) assessing whether the compound selectively modulates one or more phase transition characteristics of the condensate in the cell, wherein: the determining is by comparing the concentration of the compound at which the one or more phase transition characteristics change in the cell, as compared to a concentration of the compound at which the non-target macromolecule undergoes phase transition in the cell.

E120. The method of embodiment 119, wherein the microdroplet comprises a trigger of phase transition of a condensate that is known to modulate one or more phase transition characteristics of a condensate in vitro.

E121 . The method of embodiment 119 or 120, wherein the reference compound is a trigger of phase transition of a condensate that is known to modulate one or more phase transition characteristics of a condensate.

E122. The method of any one of embodiments 119-121 , wherein a trigger of phase transition that is known to modulate one or more phase transition characteristics of a condensate in a cell, is added to the cell.

E123. The method of any one of embodiments 120-122, wherein the trigger of phase transition is a protein, nucleic acid, salt, polyethylene glycol (PEG) or a biological mixture, optionally wherein the biological mixture is a cell lysate.

E124. The method of any one of embodiments 119-123, wherein the target macromolecule is a protein or nucleic acid.

E125. The method of embodiment 124, wherein the nucleic acid is DNA or RNA.

E126. The method of embodiment 125, wherein the RNA is mRNA, hnRNA, non-coding RNA, rRNA, tRNA, IncRNSA, or miRNA.

E127. The method of any one of embodiments 119-126, wherein the non-target macromolecule is a protein or a nucleic acid.

E128. The method of embodiment 127, wherein the non-target macromolecule is an RNA transcript, a specific RNA, a DMPK transcript, an AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

E129. The method of any one of embodiments 119-128, wherein the method comprises the step of producing the microdroplets on a microfluidic chip. E130. The method of any one of embodiments 119-129, wherein each microdroplet comprises a portion of a stock mixture comprising the target macromolecule, a portion of a stock mixture comprising the compound and a position of a stock mixture comprising a non-target macromolecule.

E131 . The method of embodiment 130, wherein each microdroplet further comprises a portion of a stock mixture comprising the trigger.

E132. The method of embodiment 130 or 131 , wherein each microdroplet is produced by automated mixing of the stock mixtures.

E133. The method of any one of embodiments 119-132, wherein the compound is added to the cell as a stock mixture comprising the compound.

E134. The method of any one of embodiments 122-133, wherein the trigger is added to the cell as a portion of a stock mixture comprising the trigger.

E135. The method of any one of embodiments 130-134, wherein:

E136. The method of any one of embodiments 130-135, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration; and the stock mixture comprising the trigger further comprises a fluorescent dye (FDtrigger) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDtrigger) correlates with the concentration of the trigger and facilitates detection of the trigger concentration.

E137. The method of embodiment 135 or 136, wherein each fluorescent dye is different from one another.

E138. The method of embodiment 136, wherein each fluorescent dye exhibits distinct excitation and emission spectra from one another.

E139. The method of any one of embodiments 135-138, wherein each fluorescent dye is selected from 7-nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUETM dye), 5-(dimethylamino)naphthalene-1 -sulfonyl (Dansyl), pyrene, 7-amino-3- {[(2,5-dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350TM), 6,8-difluoro-7-hydroxy-4-methylcoumarin (MARINA BLUETM dye), N-(2-aminoethyl)- 4-{5-[4-(dimethylamino)phenyl]-1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYLTM dye), 2, 3,5,6- Tetramethyl-1 H,7H-pyrazolo[1 ,2-a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4- (ethylamino)naphthalen-2-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUETM dye), tris(N,N-diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrolidin-1 -yl)oxy]carbonyl}piperidin- 1 -yl)-2-oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405TM), N,N-diethylethanaminium [9-{6- [(2,5-dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H- benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430TM), 1 -[({4-[(7-nitro-2,1 ,3- benzoxadiazol-4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSYTM dye), fluorescein, 2-(6- amino-3-iminio-4,5-disulfonato-3H-xanthen-9-yl)-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488TM), 2',7'-Difluoro-3',6'-dihydroxy-3H-spiro[isobenzofuran-1 ,9'-xanthen]-3-one (OREGON GREENTM 488), 1 ,3,5,7, 8-pentamethyl-4,4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPYTM 493/503), rhodamine green, 13-[2-carboxy-3,4,6-trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 - yl)oxy-6-oxohexyl]amino]-2-oxoethyl]sulfanylphenyl]-7,7,9,17,19,19-hexamethyl-2-oxa-6,20- diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa-1 (14),3,5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546TM), 2-[5-[3,3-dimethyl-5-sulfo-1 -(3-sulfopropyl)indol-1 -ium-2-yl]penta-2,4- dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5- sulfonic acid (ALEXA FLUOR 647TM), and rhodamine red;

E140. The method of any one of embodiments 1 19-139, wherein the step of determining, within each of the microdroplets, the target macromolecule is visualized by fluorescent microscopy to determine one or more phase transition characteristics of the condensate.

E141 . The method of any one of embodiments 1 19-140, wherein method identifies the compound that modulates the phase characteristics of the condensate.

E142. The method of any one of embodiments 1 19-141 , wherein the method identified the compound that selectively modulates heterotypic phase transition of the target macromolecule and the non-target macromolecule.

E143. The method of embodiment 142, wherein a finding that the target macromolecule is present within the microdroplet in discrete puncta is taken as an indication that one or more phase transition characteristics of the condensate have changed, and a finding that the target macromolecule is present in a homogenous distribution within the microdroplet, absent discrete puncta, is taken as an indication that one or more phase transition characteristics of the condensate have not changed. E144. The method of any one of embodiments 119-143, wherein steps (a) through (c) are performed on a plurality of compounds, thereby screening a compound library to identify a compound that modulates one or more phase transition characteristics of the condensate in vitro.

E145. The method of any one of embodiments 119-144, wherein steps (a’) through (b’) have been performed on a plurality of compounds, thereby screening a compound library to identify a compound that modulates the one or more phase transition characteristics of the condensate in the cell.

E146. The method of embodiment 144 or 145, wherein the screening is high-throughput screening.

E147. The method of any one of embodiments 144-146, wherein the compound library comprises from 10 to 100,000 compounds, or more.

E148. The method of any one of embodiments 119-147, wherein the concentration of the compound at which the one or more phase transition characteristics change is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet,

E149. The method of any one of embodiments 119-148, wherein the microdroplet comprises a cell lysate or cells.

E150. The method of embodiment 149, wherein the cells are from a diseased cell line.

E151 . The method of any one of embodiments 119-150, wherein the cell is a eukaryotic cell.

E152. The method of embodiment 151 , wherein the eukaryotic cell is a mammalian cell.

E153. The method of embodiment 152, wherein the mammalian cell is a human cell.

E154. The method of any one of embodiments 119-153, wherein the cell naturally expresses the target macromolecule and undergoes phase transition of the condensate or the cell is induced to express the target macromolecule.

E155. The method of any one of embodiments 120-154, wherein the trigger induces oxidative stress in the cell, thereby promoting localization of the target macromolecule to cytoplasmic stress granules.

E156. The method of embodiment 155, wherein the trigger that induces oxidative stress is sodium arsenite. E157. The method of any one of embodiments 120-156, wherein the trigger induces a change in a chemical modification state of the target macromolecule, thereby causing the target macromolecule to localize to a condensate in the cell.

E158. The method of embodiment 157, wherein the chemical modification is a post-transcriptional modification, such as phosphorylation, methylation, or acetylation.

E159. The method of embodiment 158, wherein the trigger induces a change in methylation state is adenosine dialdehyde.

E160. The method of any one of embodiments 120-159, wherein the trigger induces formation of nucleolar caps in the cell, thereby promoting localization of the target macromolecule to a nucleolar cap.

E161 . The method of embodiment 160, wherein the trigger induces formation of nucleolar caps in the cell is actinomycin D.

E162. The method of any one of embodiments 119-161 , wherein step (b’) is performed by way of fluorescent microscopy.

E163. The method of any one of embodiments 119-163, wherein the compound is identified as one that binds to, and/or modulates the activity of the target macromolecule.

E164. The method of any one of embodiments 119-163, wherein:

(a) the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that modulates heterotypic phase transition of the target macromolecule and the non-target macromolecule; or

E165. The method of any one of embodiments 119-164, wherein the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4-ALK, FUS-CHOP, YAP, TAZ or MBNL1 .

E166. A method of identifying a compound that modulates one or more phase characteristics of a condensate in a cell, the method comprising: (a’) introducing, into a cell, the compound identified as one that modulates one or more phase transition characteristics of the condensate in vitro, wherein the cell comprises the target macromolecule; and

(b’) assessing whether the compound modulates one or more phase transition characteristics of the condensate in the cell, wherein: the determining is by comparing the concentration of the compound at which the one or more phase transition characteristics change in the cell, as compared to a concentration of a reference compound at which the one or more phase transition characteristics change in the cell.

E167. The method of embodiment 166, wherein a trigger of phase transition that is known to modulate one or more phase transition characteristics of a condensate in a cell, is added to the cell.

E168. The method of embodiment 166 or 167, wherein the target macromolecule is a protein or nucleic acid.

E169. The method of embodiment 168, wherein the nucleic acid is DNA or RNA.

E170. The method of embodiment 169, wherein the RNA is mRNA, hnRNA, non-coding RNA, rRNA, tRNA, ElncRNSA, or miRNA.

E171 . The method of any one of embodiments 166-170, wherein the compound is added to the cell as a stock mixture comprising the compound.

E172. The method of any one of embodiments 167-171 , wherein the trigger is added to the cell as a portion of a stock mixture comprising the trigger.

E173. The method of embodiment 171 , wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration; and each of the target macromolecule is, independently, covalently, or noncovalently bound to a fluorescent dye (FDtarget), whereby fluorescence of one fluorescent dye (FDtarget) facilitates detection of one target macromolecule.

E174. The method of embodiment 172, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the concentration of the target macromolecule in the microdroplet; each of the target macromolecule is, independently, covalently, or noncovalently bound to a fluorescent dye (FDtarget), whereby fluorescence of each fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and the stock mixture comprising the trigger further comprises a fluorescent dye (FDtrigger) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDtrigger) correlates with the concentration of the trigger and facilitates detection of the trigger concentration.

E175. The method of embodiment 173 or 174, wherein each fluorescent dye is different from one another.

E176. The method of embodiment 175, wherein each fluorescent dye exhibits distinct excitation and emission spectra from one another.

E177. The method of any one of embodiments 173-176, wherein each fluorescent dye is selected from 7-nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUETM dye), 5-(dimethylamino)naphthalene-1 -sulfonyl (Dansyl), pyrene, 7-amino-3- {[(2,5-dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350TM), 6,8-difluoro-7-hydroxy-4-methylcoumarin (MARINA BLUETM dye), N-(2-aminoethyl)- 4-{5-[4-(dimethylamino)phenyl]-1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYLTM dye), 2, 3,5,6- Tetramethyl-1 H,7H-pyrazolo[1 ,2-a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4- (ethylamino)naphthalen-2-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUETM dye), tris(N,N-diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrolidin-1 -yl)oxy]carbonyl}piperidin- 1 -yl)-2-oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405TM), N,N-diethylethanaminium [9-{6- [(2,5-dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H- benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430TM), 1 -[({4-[(7-nitro-2,1 ,3- benzoxadiazol-4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSYTM dye), fluorescein, 2-(6- amino-3-iminio-4,5-disulfonato-3H-xanthen-9-yl)-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488TM), 2',7'-Difluoro-3',6'-dihydroxy-3H-spiro[isobenzofuran-1 ,9'-xanthen]-3-one (OREGON GREENTM 488), 1 ,3,5,7, 8-pentamethyl-4,4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPYTM 493/503), rhodamine green, 13-[2-carboxy-3,4,6-trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 - yl)oxy-6-oxohexyl]amino]-2-oxoethyl]sulfanylphenyl]-7,7,9,17,19,19-hexamethyl-2-oxa-6,20- diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa-1 (14),3,5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546TM), 2-[5-[3,3-dimethyl-5-sulfo-1 -(3-sulfopropyl)indol-1 -ium-2-yl]penta-2,4- dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5- sulfonic acid (ALEXA FLUOR 647TM), and rhodamine red;

And/or wherein one or more of the fluorescent dye is a fluorescent protein selected from GFP, mNeonGreen, mRFP, mCherry, and mPlum. E178. The method of any one of embodiments 166-177, wherein steps (a’) through (b’) have been performed on a plurality of compounds, thereby screening a compound library to identify a compound that modulates the one or more phase transition characteristics of the condensate in the cell.

E179. The method of embodiment 178, wherein the screening is high-throughput screening.

E180. The method of embodiment 178 or 179, wherein the compound library comprises from 10 to 100,000 compounds, or more.

E181 . The method of any one of embodiments 166-180, wherein the compound is identified as one that binds to and/or modulates the activity of the target macromolecule.

E182. The method of any one of embodiments 166-181 , wherein the cell is a eukaryotic cell.

E183. The method of embodiment 182, wherein the eukaryotic cell is a mammalian cell.

E184. The method of embodiment 183, wherein the mammalian cell is a human cell.

E185. The method of any one of embodiments 166-184, wherein the cell naturally expresses the target macromolecule and undergoes phase transition.

E186. The method of any one of embodiments 166-185, wherein the cell is induced to express the target macromolecule.

E187. The method of any one of embodiments 167-186, wherein the trigger induces oxidative stress in the cell, thereby promoting localization of the target macromolecule to cytoplasmic stress granules.

E188. The method of embodiment 187, wherein the trigger induces oxidative stress is sodium arsenite.

E189. The method of any one of embodiments 167-188, wherein the trigger induces a change in a chemical modification state of the target macromolecule, thereby causing the target macromolecule to localize to a condensate in the cell.

E190. The method of embodiment 189, wherein the chemical modification is a post-transcriptional modification, such as phosphorylation, methylation, or acetylation.

E191 . The method of embodiment 190, wherein the chemical modification is methylation, and wherein the trigger induces a change in methylation state is adenosine dialdehyde. E192. The method of any one of embodiments 167-191 , wherein the trigger induces formation of nucleolar caps in the cell, thereby promoting localization of the target macromolecule to a nucleolar cap.

E193. The method of embodiment 192, wherein the trigger induces formation of nucleolar caps in the cell is actinomycin D.

E194. The method of any one of embodiments 166-193 wherein step (b’) is performed by way of fluorescent microscopy.

E195. The method of any one of embodiments 166-194, wherein the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4-ALK, FUS-CHOP, YAP, TAZ or MBNL1 .

E196. A method of identifying selective condensate modulators, the method comprising contacting a condensate modulator with a composition comprising a target macromolecule and a non-target macromolecule, and measuring one or more phase transition characteristics of the condensate in a cell.

E197. The method of embodiment 196, the method comprising:

(a’) introducing, into a cell, the compound identified as one that selectively modulates one or more phase transition characteristics of the condensate, in vitro, wherein the cell comprises the target macromolecule and the non-target macromolecule; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-target), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, and

E198. The method of embodiment 197, wherein a trigger of phase transition that is known to modulate one or more phase transition characteristics of a condensate in a cell, is added to the cell.

E199. The method of embodiment 197 or 198, wherein the reference compound is a trigger of phase transition of a condensate that is known to modulate one or more phase transition characteristics of a condensate. E200. The method of embodiment 198 or 199, wherein the trigger of phase transition is a protein, nucleic acid, salt, polyethylene glycol (PEG) or a biological mixture, optionally wherein the biological mixture is a cell lysate.

E201 . The method of any one of embodiments 197-200, wherein the target macromolecule is a protein or nucleic acid.

E202. The method of embodiment 201 , wherein the nucleic acid is DNA or RNA.

E203. The method of embodiment 202, wherein the RNA is mRNA, hnRNA, non-coding RNA, rRNA, tRNA, IncRNSA, or miRNA.

E204. The method of any one of embodiments 197-203, wherein the non-target macromolecule is a protein or a nucleic acid.

E205. The method of embodiment 204, wherein the non-target macromolecule is an RNA transcript, a specific RNA, a DMPK transcript, an AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

E206. The method of any one of embodiments 197-205, wherein the compound is added to the cell as a stock mixture comprising the compound.

E207. The method of any one of embodiments 198-206, wherein the trigger is added to the cell as a portion of a stock mixture comprising the trigger.

E208. The method of embodiment 206, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration.

E209. The method of embodiment 207, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration; and the stock mixture comprising the trigger further comprises a fluorescent dye (FDtrigger) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDtrigger) correlates with the concentration of the trigger and facilitates detection of the trigger concentration.

E210. The method of embodiment 208 or 209, wherein each fluorescent dye is different from one another. E21 1 . The method of embodiment 210, wherein each fluorescent dye exhibits distinct excitation and emission spectra from one another.

E212. The method of any one of embodiments 208-21 1 , wherein each fluorescent dye is selected from 7-nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUETM dye), 5-(dimethylamino)naphthalene-1 -sulfonyl (Dansyl), pyrene, 7-amino-3- {[(2,5-dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350TM), 6,8-difluoro-7-hydroxy-4-methylcoumarin (MARINA BLUETM dye), N-(2-aminoethyl)- 4-{5-[4-(dimethylamino)phenyl]-1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYLTM dye), 2, 3,5,6- Tetramethyl-1 H,7H-pyrazolo[1 ,2-a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4- (ethylamino)naphthalen-2-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUETM dye), tris(N,N-diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrolidin-1 -yl)oxy]carbonyl}piperidin- 1 -yl)-2-oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405TM), N,N-diethylethanaminium [9-{6- [(2,5-dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H- benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430TM), 1 -[({4-[(7-nitro-2,1 ,3- benzoxadiazol-4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSYTM dye), fluorescein, 2-(6- amino-3-iminio-4,5-disulfonato-3H-xanthen-9-yl)-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488TM), 2',7'-Difluoro-3',6'-dihydroxy-3H-spiro[isobenzofuran-1 ,9'-xanthen]-3-one (OREGON GREENTM 488), 1 ,3,5,7, 8-pentamethyl-4,4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPYTM 493/503), rhodamine green, 13-[2-carboxy-3,4,6-trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 - yl)oxy-6-oxohexyl]amino]-2-oxoethyl]sulfanylphenyl]-7,7,9,17,19,19-hexamethyl-2-oxa-6,20- diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa-1 (14),3,5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546TM), 2-[5-[3,3-dimethyl-5-sulfo-1 -(3-sulfopropyl)indol-1 -ium-2-yl]penta-2,4- dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5- sulfonic acid (ALEXA FLUOR 647TM), and rhodamine red;

E213. The method of any one of embodiments 197-212, wherein method identifies the compound that modulates the phase characteristics of the condensate.

E214. The method of any one of embodiments 197-213, wherein the method identified the compound that selectively modulates heterotypic phase transition of the target macromolecule and the non-target macromolecule.

E215. The method of embodiment 213 or 214, wherein steps (a’) through (b’) have been performed on a plurality of compounds, thereby screening a compound library to identify a compound that modulates the one or more phase transition characteristics of the condensate in the cell. E216. The method of embodiment 215, wherein the screening is high-throughput screening.

E217. The method of embodiment 215 or 216, wherein the compound library comprises from 10 to 100,000 compounds, or more.

E218. The method of any one of embodiments 197-217, wherein the concentration of the compound at which the one or more phase transition characteristics change is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet, wherein the positive reference microdroplet comprises the target macromolecule and a trigger of phase transition, and the negative reference microdroplet comprises the target macromolecule.

E219. The method of any one of embodiments 197-218, wherein the microdroplet comprises a cell lysate or cells.

E220. The method of embodiment 219, wherein the cells are from a diseased cell line.

E221 . The method of any one of embodiments 197-220, wherein the cell is a eukaryotic cell.

E222. The method of embodiment 221 , wherein the eukaryotic cell is a mammalian cell.

E223. The method of embodiment 222, wherein the mammalian cell is a human cell.

E224. The method of any one of embodiments 197-223, wherein the cell naturally expresses the target macromolecule and undergoes phase transition.

E225. The method of any one of embodiments 197-224, wherein the cell is induced to express the target macromolecule.

E226. The method of any one of embodiments 198-225, wherein the trigger induces oxidative stress in the cell, thereby promoting localization of the target macromolecule to cytoplasmic stress granules.

E227. The method of embodiment 226, wherein the trigger that induces oxidative stress is sodium arsenite.

E228. The method of any one of embodiments 198-227, wherein the trigger induces a change in a chemical modification state of the target macromolecule, thereby causing the target macromolecule to localize to a condensate in the cell. E229. The method of embodiment 228, wherein the chemical modification is a post-transcriptional modification, such as phosphorylation, methylation, or acetylation.

E230. The method of embodiment 229, wherein the trigger induces a change in methylation state is adenosine dialdehyde.

E231 . The method of any one of embodiments 198-230, wherein the trigger induces formation of nucleolar caps in the cell, thereby promoting localization of the target macromolecule to a nucleolar cap.

E232. The method of embodiment 231 , wherein the trigger induces formation of nucleolar caps in the cell is actinomycin D.

E233. The method of any one of embodiments 197-232, wherein step (b’) is performed by way of fluorescent microscopy.

E234. The method of any one of embodiments 197-233, wherein the compound is identified as one that binds to, and/or modulates the activity of the target macromolecule.

E235. The method of any one of embodiments 197-234, wherein: (a) the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that modulates heterotypic phase transition of the target macromolecule and the non-target macromolecule; or

E236. The method of any one of embodiments 197-235, wherein the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4-ALK, FUS-CHOP, YAP, TAZ or MBNL1 .

E237. A method for screening a plurality of compounds to identify condensate modulators that inhibit or promote liquid-liquid phase separation (LLPS) of a target macromolecule, the method comprising: (a) producing a stream of microdroplets, wherein each of said microdroplets comprises a compound and the target macromolecule, wherein the concentration of the compound varies among the plurality of microdroplets; . (b) determining, within each of the microdroplets, whether the target macromolecule has undergone LLPS, thereby identifying the concentration of the compound at which the target macromolecule undergoes LLPS; and

(c) determining whether the compound promotes or inhibits LLPS, wherein: the determining is performed by comparing the concentration of the compound at which the target macromolecule undergoes LLPS as compared to a concentration of a reference compound at which the target macromolecule undergoes LLPS, the compound promotes LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is less than the concentration of the reference compound at which the target macromolecule undergoes LLPS, and the compound inhibits LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is greater than the concentration of the reference compound at which the target macromolecule undergoes LLPS.

E238. The method of embodiment 237, wherein the microdroplet comprises a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule in vitro.

E239. The method of any one of embodiments 237-238, wherein the reference compound is a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule.

E240. The method of any one of embodiments 237-239, wherein the microdroplet comprises a cell lysate.

E241 . The method of any one of embodiments 238-240, wherein the trigger of LLPS is a protein, nucleic acid, salt, polyethylene glycol (PEG).

E242. The method of embodiment 241 , wherein the polyethylene glycol (PEG) has an average molecular weight of from 600 Da to 20 kDa or about 10 kDa.

E243. The method of any one of embodiments 237-242, wherein the target macromolecule is a protein or nucleic acid.

E244. The method of embodiment 243, wherein the nucleic acid is DNA or RNA.

E245. The method of embodiment 244, wherein the RNA is mRNA, hnRNA, non-coding RNA, rRNA, tRNA, IncRNSA, or miRNA.

E246. The method of any one of embodiments 237-245, wherein the method comprises the step of producing the microdroplets on a microfluidic chip. E247. The method of any one of embodiments 237-246, wherein each microdroplet comprises a portion of a stock mixture comprising the target macromolecule and a portion of a stock mixture comprising the compound.

E248. The method of embodiment 247, wherein each microdroplet further comprises a portion of a stock mixture comprising the trigger.

E249. The method of any one of embodiments 247-248, wherein each microdroplet is produced by automated mixing of the stock mixtures.

E250. The method of any one of embodiments 247-249, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration; and each of the target macromolecule is, independently, covalently or noncovalently bound to a fluorescent dye (FDtarget), whereby fluorescence of one fluorescent dye (FDtarget) facilitates detection of one target macromolecule.

E251 . The method of any one of embodiment 247-250, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration; each of the target macromolecule is, independently, covalently, or noncovalently bound to a fluorescent dye (FDtarget), whereby fluorescence of each fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and the stock mixture comprising the trigger further comprises a fluorescent dye (FDtrigger) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDtrigger) correlates with the concentration of the trigger and facilitates detection of the trigger concentration.

E252. The method of embodiment 250 or 251 , wherein each fluorescent dye is different from one another.

E253. The method of embodiment 252, wherein each fluorescent dye exhibits distinct excitation and emission spectra from one another.

E254. The method of any one of embodiments 250-253, wherein each fluorescent dye is selected from 7-nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUETM dye), 5-(dimethylamino)naphthalene-1 -sulfonyl (Dansyl), pyrene, 7-amino-3- {[(2,5-dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350TM), 6,8-difluoro-7-hydroxy-4-methylcoumarin (MARINA BLUETM dye), N-(2-aminoethyl)- 4-{5-[4-(dimethylamino)phenyl]-1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYLTM dye), 2, 3,5,6- Tetramethyl-1 H,7H-pyrazolo[1 ,2-a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4- (ethylamino)naphthalen-2-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUETM dye), tris(N,N-diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrolidin-1 -yl)oxy]carbonyl}piperidin- 1 -yl)-2-oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405TM), N,N-diethylethanaminium [9-{6- [(2,5-dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H- benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430TM), 1 -[({4-[(7-nitro-2,1 ,3- benzoxadiazol-4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSYTM dye), fluorescein, 2-(6- amino-3-iminio-4,5-disulfonato-3H-xanthen-9-yl)-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488TM), 2',7'-Difluoro-3',6'-dihydroxy-3H-spiro[isobenzofuran-1 ,9'-xanthen]-3-one (OREGON GREENTM 488), 1 ,3,5,7, 8-pentamethyl-4,4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPYTM 493/503), rhodamine green, 13-[2-carboxy-3,4,6-trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 - yl)oxy-6-oxohexyl]amino]-2-oxoethyl]sulfanylphenyl]-7,7,9,17,19,19-hexamethyl-2-oxa-6,20- diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa-1 (14),3,5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546TM), 2-[5-[3,3-dimethyl-5-sulfo-1 -(3-sulfopropyl)indol-1 -ium-2-yl]penta-2,4- dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5- sulfonic acid (ALEXA FLUOR 647TM), and rhodamine red;

E255. The method of any one of embodiments 237-254, wherein the step of determining, within each of the microdroplets, the target macromolecule is visualized by fluorescent microscopy to determine if the target macromolecule has undergone LLPS.

E256. The method of embodiment 255, wherein a finding that the target macromolecule is present within the microdroplet in discrete puncta is taken as an indication that the target macromolecule has undergone LLPS, and a finding that the target macromolecule is present in a homogenous distribution within the microdroplet, absent discrete puncta, is taken as an indication that the target macromolecule has not undergone LLPS.

E257. The method of any one of embodiments 237-256, wherein steps (a) through (c) are performed on a plurality of compounds, thereby screening a compound library to identify a compound that inhibits or promotes LLPS of the target macromolecule in vitro.

E258. The method of embodiment 257, wherein the screening is high-throughput screening.

E259. The method of embodiment 257 or 258, wherein the compound library comprises from 10 to 100,000 compounds, or more. E260. The method of any one of embodiments 237-259, wherein the concentration of the compound at which the target macromolecule undergoes LLPS is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet, wherein the positive reference microdroplet comprises the target macromolecule and a trigger of LLPS, and the negative reference microdroplet comprises the target macromolecule.

E261 . The method of any one of embodiments 237-260, wherein the compound is identified as one that binds to and/or modulates the activity of the target macromolecule.

E262. The method of any one of embodiments 237-261 , wherein the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4-ALK, FUS-CHOP, YAP, TAZ or MBNL1 .

E263. The method of any one of embodiments 237-262, wherein the microdroplet comprises a cell lysate or cells.

E264. The method of any one of embodiments 237-263, wherein the cells are from a diseased cell line.

E265. The method of any one of embodiments 237-264, wherein the microdroplet comprises a non-target macromolecule; optionally wherein the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that modulates phase transition characteristics of a condensate system that comprises the target macromolecule.

E266. The method of embodiment 265, wherein the non-target macromolecule is an RNA transcript.

E267. The method of embodiment 266, wherein the non-target macromolecule is a specific RNA, a DMPK transcript or AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

E268. A method of determining whether a compound inhibits or promotes LLPS of a target macromolecule, the method comprising:

(b) determining, within each of the microdroplets, whether the target macromolecule has undergone LLPS, thereby identifying the concentration of the compound at which the target macromolecule undergoes LLPS;

(c) determining whether the compound promotes or inhibits LLPS, wherein: the determining is by comparing the concentration of the compound at which the target macromolecule undergoes LLPS as compared to a concentration of a reference compound at which the target macromolecule undergoes LLPS, the compound promotes LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is less than the concentration of the reference compound at which the target macromolecule undergoes LLPS, and the compound inhibits LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is greater than the concentration of the reference compound at which the target macromolecule undergoes LLPS,

(a’) introducing, into a cell, the compound identified in (c) as one that promotes or inhibits LLPS of the target macromolecule in vitro, wherein the cell comprises the target macromolecule; and

(b’) assessing whether the compound promotes or inhibits LLPS of the target macromolecule in the cell, wherein: the determining is by comparing the concentration of the compound at which the target macromolecule undergoes LLPS in the cell, as compared to a concentration of a reference compound at which the target macromolecule undergoes LLPS in the cell, and wherein the compound promotes LLPS in the cell when the concentration of the compound at which the target macromolecule undergoes LLPS in the cell is less than the concentration of the reference compound at which the target macromolecule undergoes LLPS in the cell, and the compound inhibits LLPS in the cell when the concentration of the compound at which the target macromolecule undergoes LLPS in the cell is greater than the concentration of the reference compound at which the target macromolecule undergoes LLPS in the cell.

E269. The method of embodiment 268, wherein the microdroplet comprises a cell lysate.

E270. The method of embodiment 268 or 269, wherein the microdroplet comprises a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule in vitro.

E271 . The method of any one of embodiments 268-270, wherein a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule in a cell, is added to the cell.

E272. The method of any one of embodiments 268-271 , wherein the reference compound is a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule.

E273. The method of any one of embodiments 269-272, wherein the trigger of LLPS is a protein, nucleic acid, salt, polyethylene glycol (PEG), or a biological mixture, optionally wherein the biological mixture is a cell lysate. E274. The method of embodiment 273, wherein the PEG has an average molecular weight of from 600 Da to 20 kDa, or about 10 kDa.

E275. The method of any one of embodiments 268-274, wherein the target macromolecule is a protein or nucleic acid.

E276. The method of embodiment 275, wherein the nucleic acid is DNA or RNA.

E277. The method of embodiment 276, wherein the RNA is mRNA, hnRNA, non-coding RNA, rRNA, tRNA, IncRNSA, or miRNA.

E278. The method of any one of embodiments 268-277, wherein the method comprises the step of producing the microdroplets on a microfluidic chip.

E279. The method of any one of embodiments 268-278, wherein each microdroplet comprises a portion of a stock mixture comprising the target macromolecule and a portion of a stock mixture comprising the compound.

E280. The method of embodiment 279, wherein each microdroplet further comprises a portion of a stock mixture comprising the trigger.

E281 . The method of embodiment 279 or 280, wherein each microdroplet is produced by automated mixing of the stock mixtures.

E282. The method of any one of embodiments 268-281 , wherein the compound is added to the cell as a stock mixture comprising the compound.

E283. The method of any one of embodiments 269-282, wherein the trigger is added to the cell as a portion of a stock mixture comprising the trigger.

E284. The method of any one of embodiments 279-283, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration; and each of the target macromolecule is, independently, covalently, or noncovalently bound to a fluorescent dye (FDtarget), whereby fluorescence of one fluorescent dye (FDtarget) facilitates detection of one target macromolecule.

E285. The method of any one of embodiment 279-284, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration; each of the target macromolecule is, independently, covalently, or noncovalently bound to a fluorescent dye (FDtarget), whereby fluorescence of each fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and the stock mixture comprising the trigger further comprises a fluorescent dye (FDtrigger) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDtrigger) correlates with the concentration of the trigger and facilitates detection of the trigger concentration.

E286. The method of embodiment 284 or 285, wherein each fluorescent dye is different from one another.

E287. The method of embodiment 286, wherein each fluorescent dye exhibits distinct excitation and emission spectra from one another.

E288. The method of any one of embodiments 284-287, wherein each fluorescent dye is selected from 7-nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUETM dye), 5-(dimethylamino)naphthalene-1 -sulfonyl (Dansyl), pyrene, 7-amino-3- {[(2,5-dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350TM), 6,8-difluoro-7-hydroxy-4-methylcoumarin (MARINA BLUETM dye), N-(2-aminoethyl)- 4-{5-[4-(dimethylamino)phenyl]-1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYLTM dye), 2, 3,5,6- Tetramethyl-1 H,7H-pyrazolo[1 ,2-a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4- (ethylamino)naphthalen-2-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUETM dye), tris(N,N-diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrolidin-1 -yl)oxy]carbonyl}piperidin- 1 -yl)-2-oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405TM), N,N-diethylethanaminium [9-{6- [(2,5-dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H- benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430TM), 1 -[({4-[(7-nitro-2,1 ,3- benzoxadiazol-4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSYTM dye), fluorescein, 2-(6- amino-3-iminio-4,5-disulfonato-3H-xanthen-9-yl)-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488TM), 2',7'-Difluoro-3',6'-dihydroxy-3H-spiro[isobenzofuran-1 ,9'-xanthen]-3-one (OREGON GREENTM 488), 1 ,3,5,7, 8-pentamethyl-4,4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPYTM 493/503), rhodamine green, 13-[2-carboxy-3,4,6-trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 - yl)oxy-6-oxohexyl]amino]-2-oxoethyl]sulfanylphenyl]-7,7,9,17,19,19-hexamethyl-2-oxa-6,20- diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa-1 (14),3,5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546TM), 2-[5-[3,3-dimethyl-5-sulfo-1 -(3-sulfopropyl)indol-1 -ium-2-yl]penta-2,4- dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5- sulfonic acid (ALEXA FLUOR 647TM), and rhodamine red; And/or wherein one or more of the fluorescent dye is a fluorescent protein selected from GFP, mNeonGreen, mRFP, mCherry, and mPlum.

E289. The method of any one of embodiments 268-288, wherein the step of determining, within each of the microdroplets, the target macromolecule is visualized by fluorescent microscopy to determine if the target macromolecule has undergone LLPS.

E290. The method of embodiment 289, wherein a finding that the target macromolecule is present within the microdroplet in discrete puncta is taken as an indication that the target macromolecule has undergone LLPS, and a finding that the target macromolecule is present in a homogenous distribution within the microdroplet, absent discrete puncta, is taken as an indication that the target macromolecule has not undergone LLPS.

E291 . The method of any one of embodiments 268-290, wherein steps (a) through (c) are performed on a plurality of compounds, thereby screening a compound library to identify a compound that inhibits or promotes LLPS of the target macromolecule in vitro.

E292. The method of any one of embodiments 268-291 , wherein steps (a’) through (b’) have been performed on a plurality of compounds, thereby screening a compound library to identify a compound that inhibits or promotes LLPS of the target macromolecule in the cell.

E293. The method of embodiment 291 or 292, wherein the screening is high-throughput screening.

E294. The method of any one of embodiments 291 -293, wherein the compound library comprises from 10 to 100,000 compounds, or more.

E295. The method of any one of embodiments 268-294, wherein the concentration of the compound at which the condensate undergoes phase transition is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet, wherein the positive reference microdroplet comprises the target macromolecule and a trigger of phase transition, and the negative reference microdroplet comprises the target macromolecule.

E296. The method of any one of embodiments 268-295, wherein the microdroplet comprises a cell from a diseased cell line.

E297. The method of any one of embodiments 268-296, wherein the microdroplet comprises a non-target macromolecule; optionally wherein the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that phase separation of a condensate system that comprises the target macromolecule.

E298. The method of embodiment 297, wherein the non-target macromolecule is an RNA transcript.

E299. The method of embodiment 298, wherein the non-target macromolecule is a specific RNA, a DMPK transcript or AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

E300. The method of any one of embodiments 268-299, wherein the cell is a eukaryotic cell.

E301 . The method of embodiment 300, wherein the eukaryotic cell is a mammalian cell.

E302. The method of embodiment 301 , wherein the mammalian cell is a human cell.

E303. The method of any one of embodiments 268-302, wherein the cell naturally expresses the target macromolecule and undergoes LLPS.

E304. The method of any one of embodiments 268-303, wherein the cell is induced to express the target macromolecule.

E305. The method of any one of embodiments 269-304, wherein the trigger induces oxidative stress in the cell, thereby promoting localization of the target macromolecule to cytoplasmic stress granules.

E306. The method of embodiment 305, wherein the trigger induces oxidative stress is sodium arsenite.

E307. The method of any one of embodiments 269-306, wherein the trigger induces a change in a chemical modification state of the target macromolecule, thereby causing the target macromolecule to localize to a condensate in the cell.

E308. The method of embodiment 307, wherein the chemical modification is a post-translation modification, such as phosphorylation, methylation, or acetylation.

E309. The method of embodiment 308, wherein the chemical modification is methylation, and wherein the trigger induces a change in methylation state is adenosine dialdehyde.

E310. The method of any one of embodiments 269-309, wherein the trigger induces formation of nucleolar caps in the cell, thereby promoting localization of the target macromolecule to a nucleolar cap. E311 . The method of embodiment 310, wherein the trigger induces formation of nucleolar caps in the cell is actinomycin D.

E312. The method of any one of embodiments 268-311 wherein step (b’) is performed by way of fluorescent microscopy.

E313. The method of any one of embodiments 268-312, wherein the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4-ALK, FUS-CHOP, YAP, TAZ or MBNL1 .

E314. A method of determining whether a compound selectively inhibits or promotes LLPS of a target macromolecule, the method comprising:

(b) determining, within each of the microdroplets, whether the target macromolecule has undergone LLPS, and whether the non-target macromolecule has undergone LLPS, thereby identifying the concentration of the compound at which the target macromolecule undergo LLPS and the concentration of the compound at which the non-target macromolecule undergoes LLPS;

(c) determining whether the compound selectively promotes or inhibits LLPS of the target macromolecule, wherein: the determining is by comparing the concentration of the compound at which the target macromolecule undergoes LLPS as compared to a concentration of at which the non-target macromolecule undergoes LLPS, the compound promotes LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is less than the concentration of the compound at which the non- target macromolecule undergoes LLPS, and the compound inhibits LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is greater than the concentration of the compound at which the non- target macromolecule undergoes LLPS.

E315. The method of embodiment 314, wherein the microdroplet comprises a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule in vitro. E316. The method of embodiment 314 or 315, wherein the reference compound is a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule.

E317. The method of any one of embodiments 314-316, wherein a trigger of phase transition that is known to modulate one or more phase transition characteristics of a condensate in a cell, is added to the cell.

E318. The method of any one of embodiments 315-317, wherein the trigger of LLPS is a protein, nucleic acid, salt, polyethylene glycol (PEG), or a biological mixture, optionally wherein the biological mixture is a cell lysate.

E319. The method of embodiment 318, wherein the PEG has an average molecular weight of from 600 Da to 20 kDa.

E320. The method of embodiment 318 or 319, wherein the PEG has an average molecular weight of about 10 kDa.

E321 . The method of any one of embodiments 314-320, wherein the target macromolecule is a protein or nucleic acid.

E322. The method of embodiment 321 , wherein the nucleic acid is DNA or RNA.

E323. The method of embodiment 322, wherein the RNA is mRNA, hnRNA, non-coding RNA, rRNA, tRNA, IncRNSA, or miRNA.

E324. The method of any one of embodiments 314-323, wherein the non-target macromolecule is a protein or a nucleic acid.

E325. The method of embodiment 324, wherein the non-target macromolecule is an RNA transcript, a specific RNA, a DMPK transcript, an AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

E326. The method of any one of embodiments 314-325, wherein the method comprises the step of producing the microdroplets on a microfluidic chip.

E327. The method of any one of embodiments 314-326, wherein each microdroplet comprises a portion of a stock mixture comprising the target macromolecule, a portion of a stock mixture comprising the compound and a position of a stock mixture comprising a non-target macromolecule. E328. The method of any one of the embodiments 314-327, wherein each microdroplet further comprises a portion of a stock mixture comprising the trigger.

E329. The method of embodiment 327 or 328, wherein each microdroplet is produced by automated mixing of the stock mixtures.

E330. The method of any one of embodiments 327 or 329, wherein:

E331 . The method of embodiment 328 or 329, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration; and the stock mixture comprising the trigger further comprises a fluorescent dye (FDtrigger) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDtrigger) correlates with the concentration of the trigger and facilitates detection of the trigger concentration.

E332. The method of embodiment 330 or 331 , wherein each fluorescent dye is different from one another.

E333. The method of embodiment 332, wherein each fluorescent dye exhibits distinct excitation and emission spectra from one another.

E334. The method of any one of embodiments 330-333, wherein each fluorescent dye is selected from 7-nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUETM dye), 5-(dimethylamino)naphthalene-1 -sulfonyl (Dansyl), pyrene, 7-amino-3- {[(2,5-dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350TM), 6,8-difluoro-7-hydroxy-4-methylcoumarin (MARINA BLUETM dye), N-(2-aminoethyl)- 4-{5-[4-(dimethylamino)phenyl]-1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYLTM dye), 2, 3,5,6- Tetramethyl-1 H,7H-pyrazolo[1 ,2-a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4- (ethylamino)naphthalen-2-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUETM dye), tris(N,N-diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrolidin-1 -yl)oxy]carbonyl}piperidin- 1 -yl)-2-oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405TM), N,N-diethylethanaminium [9-{6- [(2,5-dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H- benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430TM), 1 -[({4-[(7-nitro-2,1 ,3- benzoxadiazol-4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSYTM dye), fluorescein, 2-(6- amino-3-iminio-4,5-disulfonato-3H-xanthen-9-yl)-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488TM), 2',7'-Difluoro-3',6'-dihydroxy-3H-spiro[isobenzofuran-1 ,9'-xanthen]-3-one (OREGON GREENTM 488), 1 ,3,5,7, 8-pentamethyl-4,4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPYTM 493/503), rhodamine green, 13-[2-carboxy-3,4,6-trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 - yl)oxy-6-oxohexyl]amino]-2-oxoethyl]sulfanylphenyl]-7,7,9,17,19,19-hexamethyl-2-oxa-6,20- diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa-1 (14),3,5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546TM), 2-[5-[3,3-dimethyl-5-sulfo-1 -(3-sulfopropyl)indol-1 -ium-2-yl]penta-2,4- dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5- sulfonic acid (ALEXA FLUOR 647TM), and rhodamine red;

E335. The method of any one of embodiments 314-334, wherein the step of determining, within each of the microdroplets, the target macromolecule is visualized by fluorescent microscopy to determine if the target macromolecule has undergone LLPS.

E336. The method of any one of embodiments 314-335, wherein method identifies the compound that selectively promotes or inhibits homotypic LLPS of the target molecule.

E337. The method of any one of embodiments 314-336, wherein the method identified the compound that selectively promotes or inhibits heterotypic LLPS of the target macromolecule and the non-target macromolecule.

E338. The method of any one of embodiments 314-337, wherein a finding that the target macromolecule is present within the microdroplet in discrete puncta is taken as an indication that the target macromolecule has undergone LLPS, and a finding that the target macromolecule is present in a homogenous distribution within the microdroplet, absent discrete puncta, is taken as an indication that the target macromolecule has not undergone LLPS.

E339. The method of any one of embodiments 314-338, wherein steps (a) through (c) are performed on a plurality of compounds, thereby screening a compound library to identify a compound that inhibits or promotes LLPS of the target macromolecule in vitro.

E340. The method of embodiment 339, wherein the screening is high-throughput screening.

E341 . The method of any one of embodiments 339-340, wherein the compound library comprises from 10 to 100,000 compounds, or more.

E342. The method of any one of embodiments 314-341 , wherein the concentration of the compound at which the target macromolecule undergoes LLPS is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet, wherein the positive reference microdroplet comprises the target macromolecule and a trigger of LLPS, and the negative reference microdroplet comprises the target macromolecule.

E343. The method of any one of embodiments 314-342, wherein the microdroplet comprises a cell lysate or cells.

E344. The method of any one of embodiments 314-343, wherein the cells are from a diseased cell line.

E345. The method of any one of embodiments 314-344, wherein the microdroplet comprises a non-target macromolecule.

E346. The method of embodiment 345, wherein the non-target macromolecule is an RNA transcript.

E347. The method of embodiment 346, wherein the non-target macromolecule is a specific RNA, a DMPK transcript or AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

E348. The method of any one of embodiments 314-347, wherein the cell is a eukaryotic cell.

E349. The method of embodiment 348, wherein the eukaryotic cell is a mammalian cell.

E350. The method of embodiment 349, wherein the mammalian cell is a human cell.

E351 . The method of any one of embodiments 314-350, wherein the cell naturally expresses the target macromolecule and undergoes LLPS.

E352. The method of any one of embodiments 314-351 , wherein the cell is induced to express the target macromolecule.

E353. The method of any one of embodiments 314-352, wherein the trigger induces oxidative stress in the cell, thereby promoting localization of the target macromolecule to cytoplasmic stress granules.

E354. The method of embodiment 353, wherein the trigger that induces oxidative stress is sodium arsenite.

E355. The method of any one of embodiments 315-354, wherein the trigger induces a change in a chemical modification state of the target macromolecule, thereby causing the target macromolecule to localize to a condensate in the cell. E356. The method of embodiment 355, wherein the chemical modification is a post-transcriptional modification, such as phosphorylation, methylation, or acetylation.

E357. The method of embodiment 356, wherein the trigger induces a change in methylation state is adenosine dialdehyde.

E358. The method of any one of embodiments 315-357, wherein the trigger induces formation of nucleolar caps in the cell, thereby promoting localization of the target macromolecule to a nucleolar cap.

E359. The method of embodiment 358, wherein the trigger induces formation of nucleolar caps in the cell is actinomycin D.

E360. The method of any one of embodiments 314-359, wherein the compound is identified as one that binds to, and/or modulates the activity of the target macromolecule.

E361 . The method of any one of embodiments 314-360, wherein:

(a) the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that modulates heterotypic LLPS of the target macromolecule and the non-target macromolecule; or

(b) the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that phase separation of a condensate system that comprises the target macromolecule.

E362. The method of any one of embodiments 314-361 , wherein the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4-ALK, FUS-CHOP, YAP, TAZ or MBNL1 .

E363. A method of determining whether a compound selectively inhibits or promotes LLPS of a target macromolecule, the method comprising:

(a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, a non-target macromolecule and wherein the concentration of the compound varies among the plurality of microdroplets; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-target), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, (b) determining, within each of the microdroplets, whether the target macromolecule has undergone LLPS, and whether the non-target macromolecule has undergone LLPS, thereby identifying the concentration of the compound at which the target macromolecule undergo LLPS and the concentration of the compound at which the non-target macromolecule undergoes LLPS;

(c) determining whether the compound selectively promotes or inhibits LLPS of the target macromolecule, wherein: the determining is by comparing the concentration of the compound at which the target macromolecule undergoes LLPS as compared to a concentration of at which the non-target macromolecule undergoes LLPS, the compound promotes LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is less than the concentration of the compound at which the non- target macromolecule undergoes LLPS, and the compound inhibits LLPS when the concentration of the compound at which the target macromolecule undergo LLPS is greater than the concentration of the compound at which the non- target macromolecule undergoes LLPS,

(a’) introducing, into a cell, the compound identified in (c) as one that selectively promotes or inhibits LLPS of the target macromolecule in vitro, wherein the cell comprises the target macromolecule and the non-target macromolecule; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-target), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, and

(b’) assessing whether the compound selectively promotes or inhibits LLPS of the target macromolecule in the cell, wherein: the determining is by comparing the concentration of the compound at which the target macromolecule undergoes LLPS in the cell, as compared to a concentration of the compound at which the non-target macromolecule undergoes LLPS in the cell, the compound promotes LLPS in the cell when the concentration of the compound at which target macromolecule undergoes LLPS in the cell is less than the concentration of the compound at which the non-target macromolecule undergoes LLPS in the cell, and the compound inhibits LLPS in the cell when the concentration of the compound at which the target macromolecule undergoes LLPS in the cell is greater than the concentration of the compound at which the non-target macromolecule undergoes LLPS in the cell.

E364. The method of embodiment 363, wherein the microdroplet comprises a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule in vitro. E365. The method of any one of embodiments 363-364, wherein the reference compound is a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule.

E366. The method of any one of embodiments 363-365, wherein a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule in a cell, is added to the cell.

E367. The method of any one of embodiments 364-366, wherein the trigger of LLPS is a protein, nucleic acid, salt, or polyethylene glycol (PEG), optionally wherein the PEG has an average molecular weight of from 600 Da to 20 kDa, optionally wherein the PEG has an average molecular weight of about 10 kDa.

E368. The method of any one of embodiments 363-367, wherein the target macromolecule is a protein or nucleic acid.

E369. The method of embodiment 368, wherein the nucleic acid is DNA or RNA.

E370. The method of embodiment 369, wherein the RNA is mRNA, hnRNA, non-coding RNA, rRNA, tRNA, IncRNSA, or miRNA.

E371 . The method of any one of embodiments 363-370, wherein the non-target macromolecule is a protein or a nucleic acid.

E372. The method of embodiment 371 , wherein the non-target macromolecule is an RNA transcript, a specific RNA, a DMPK transcript, an AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

E373. The method of any one of embodiments 363-372, wherein the method comprises the step of producing the microdroplets on a microfluidic chip.

E374. The method of any one of embodiments 363-373, wherein the compound is added to the cell as a stock mixture comprising the compound.

E375. The method of any one of embodiments 366-374, wherein the trigger is added to the cell as a portion of a stock mixture comprising the trigger.

E376. The method of any one of embodiments 363-375, wherein each microdroplet comprises a portion of a stock mixture comprising the target macromolecule, a portion of a stock mixture comprising the compound and a position of a stock mixture comprising a non-target macromolecule. E377. The method of any one of the embodiments 364-376, wherein each microdroplet further comprises a portion of a stock mixture comprising the trigger.

E378. The method of embodiment 376 or 377, wherein each microdroplet is produced by automated mixing of the stock mixtures.

E379. The method of any one of embodiments 363-378, wherein the compound is added to the cell as a stock mixture comprising the compound.

E380. The method of any one of embodiments 366-379, wherein the trigger is added to the cell as a portion of a stock mixture comprising the trigger.

E381 . The method of any one of embodiments 376-380, wherein:

E382. The method of embodiment 377-381 , wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration; and the stock mixture comprising the trigger further comprises a fluorescent dye (FDtrigger) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDtrigger) correlates with the concentration of the trigger and facilitates detection of the trigger concentration.

E383. The method of embodiment 381 or 382, wherein each fluorescent dye is different from one another.

E384. The method of embodiment 383, wherein each fluorescent dye exhibits distinct excitation and emission spectra from one another.

E385. The method of any one of embodiments 381 -384, wherein each fluorescent dye is selected from 7-nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUETM dye), 5-(dimethylamino)naphthalene-1 -sulfonyl (Dansyl), pyrene, 7-amino-3- {[(2,5-dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350TM), 6,8-difluoro-7-hydroxy-4-methylcoumarin (MARINA BLUETM dye), N-(2-aminoethyl)- 4-{5-[4-(dimethylamino)phenyl]-1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYLTM dye), 2, 3,5,6- Tetramethyl-1 H,7H-pyrazolo[1 ,2-a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4- (ethylamino)naphthalen-2-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUETM dye), tris(N,N-diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrolidin-1 -yl)oxy]carbonyl}piperidin- 1 -yl)-2-oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405TM), N,N-diethylethanaminium [9-{6- [(2,5-dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H- benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430TM), 1 -[({4-[(7-nitro-2,1 ,3- benzoxadiazol-4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSYTM dye), fluorescein, 2-(6- amino-3-iminio-4,5-disulfonato-3H-xanthen-9-yl)-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488TM), 2',7'-Difluoro-3',6'-dihydroxy-3H-spiro[isobenzofuran-1 ,9'-xanthen]-3-one (OREGON GREENTM 488), 1 ,3,5,7, 8-pentamethyl-4,4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPYTM 493/503), rhodamine green, 13-[2-carboxy-3,4,6-trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 - yl)oxy-6-oxohexyl]amino]-2-oxoethyl]sulfanylphenyl]-7,7,9,17,19,19-hexamethyl-2-oxa-6,20- diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa-1 (14),3,5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546TM), 2-[5-[3,3-dimethyl-5-sulfo-1 -(3-sulfopropyl)indol-1 -ium-2-yl]penta-2,4- dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5- sulfonic acid (ALEXA FLUOR 647TM), and rhodamine red;

E386. The method of any one of embodiments 363-385, wherein the step of determining, within each of the microdroplets, the target macromolecule is visualized by fluorescent microscopy to determine if the target macromolecule has undergone LLPS.

E387. The method of any one of embodiments 363-386, wherein method identifies the compound that selectively promotes or inhibits homotypic LLPS of the target molecule.

E388. The method of any one of embodiments 363-387, wherein the method identified the compound that selectively promotes or inhibits heterotypic LLPS of the target macromolecule and the non-target macromolecule.

E389. The method of any one of embodiments 363-388, wherein a finding that the target macromolecule is present within the microdroplet in discrete puncta is taken as an indication that the target macromolecule has undergone LLPS, and a finding that the target macromolecule is present in a homogenous distribution within the microdroplet, absent discrete puncta, is taken as an indication that the target macromolecule has not undergone LLPS.

E390. The method of any one of embodiments 363-389, wherein steps (a) through (c) are performed on a plurality of compounds, thereby screening a compound library to identify a compound that inhibits or promotes LLPS of the target macromolecule in vitro. E391 . The method of any one of embodiments 363-390, wherein steps (a’) through (b’) have been performed on a plurality of compounds, thereby screening a compound library to identify a compound that inhibits or promotes LLPS of the target macromolecule in the cell.

E392. The method of embodiment 390 or 391 , wherein the screening is high-throughput screening.

E393. The method of any one of embodiments 390-392, wherein the compound library comprises from 10 to 100,000 compounds, or more.

E394. The method of any one of embodiments 363-393, wherein the concentration of the compound at which the target macromolecule undergoes LLPS is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet,

Wherein the positive reference microdroplet comprises the target macromolecule and a trigger of LLPS, and the negative reference microdroplet comprises the target macromolecule.

E395. The method of any one of embodiments 363-394, wherein the microdroplet comprises a cell lysate or cells.

E396. The method of embodiment 395, wherein the cells are from a diseased cell line.

E397. The method of any one of embodiments 363-396, wherein the microdroplet comprises a non-target macromolecule.

E398. The method of embodiment 397, wherein the non-target macromolecule is an RNA transcript.

E399. The method of embodiment 398, wherein the non-target macromolecule is a specific RNA, a DMPK transcript or AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

E400. The method of any one of embodiments 363-399, wherein the cell is a eukaryotic cell.

E401 . The method of embodiment 400, wherein the eukaryotic cell is a mammalian cell.

E402. The method of embodiment 401 , wherein the mammalian cell is a human cell.

E403. The method of any one of embodiments 363-402, wherein the cell naturally expresses the target macromolecule and undergoes LLPS.

E404. The method of any one of embodiments 363-403, wherein the cell is induced to express the target macromolecule. E405. The method of any one of embodiments 364-404, wherein the trigger induces oxidative stress in the cell, thereby promoting localization of the target macromolecule to cytoplasmic stress granules.

E406. The method of embodiment 405, wherein the trigger that induces oxidative stress is sodium arsenite.

E407. The method of any one of embodiments 364-406, wherein the trigger induces a change in a chemical modification state of the target macromolecule, thereby causing the target macromolecule to localize to a condensate in the cell.

E408. The method of embodiment 407, wherein the chemical modification is a post-transcriptional modification, such as phosphorylation, methylation, or acetylation.

E409. The method of embodiment 408, wherein the trigger induces a change in methylation state is adenosine dialdehyde.

E410. The method of any one of embodiments 364-409, wherein the trigger induces formation of nucleolar caps in the cell, thereby promoting localization of the target macromolecule to a nucleolar cap.

E411 . The method of embodiment 410, wherein the trigger induces formation of nucleolar caps in the cell is actinomycin D.

E412. The method of any one of embodiments 363-411 wherein step (b’) is performed by way of fluorescent microscopy.

E413. The method of any one of embodiments 363-412, wherein the compound is identified as one that binds to, and/or modulates the activity of the target macromolecule.

E414. The method of any one of embodiments 363-413, wherein:

(b) the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that phase separation of a condensate system that comprises the target macromolecule. E415. The method of any one of embodiments 363-414, wherein the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4-ALK, FUS-CHOP, YAP, TAZ or MBNL1 .

E416. A method of determining whether a compound inhibits or promotes LLPS of a target macromolecule in a cell , the method comprising:

(a’) introducing, into a cell, the compound identified as one that promotes or inhibits LLPS of the target macromolecule in vitro, wherein the cell comprises the target macromolecule; and

E417. The method of embodiment 416, wherein a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule in a cell, is added to the cell.

E418. The method of embodiment 416 or 417, wherein the reference compound is a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule.

E419. The method of any one of embodiments 416-418, wherein the trigger of LLPS is a protein, nucleic acid, salt, polyethylene glycol (PEG), or a biological mixture, optionally wherein the biological mixture is a cell lysate.

E420. The method of embodiment 419, wherein the PEG has an average molecular weight of from 600 Da to 20 kDa, or about 10 kDa.

E421 . The method of any one of embodiments 416-420, wherein the target macromolecule is a protein or nucleic acid.

E422. The method of embodiment 421 , wherein the nucleic acid is DNA or RNA.

E423. The method of embodiment 422, wherein the RNA is mRNA, hnRNA, non-coding RNA, rRNA, tRNA, IncRNSA, or miRNA. E424. The method of any one of embodiments 416-423, wherein the compound is added to the cell as a stock mixture comprising the compound.

E425. The method of any one of embodiments 417-424, wherein the trigger is added to the cell as a portion of a stock mixture comprising the trigger.

E426. The method of embodiment 424 or 425, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration; and each of the target macromolecule is, independently, covalently or noncovalently bound to a fluorescent dye (FDtarget), whereby fluorescence of one fluorescent dye (FDtarget) facilitates detection of one target macromolecule.

E427. The method of embodiment 426, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration; each of the target macromolecule is, independently, covalently, or noncovalently bound to a fluorescent dye (FDtarget), whereby fluorescence of each fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and the stock mixture comprising the trigger further comprises a fluorescent dye (FDtrigger) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDtrigger) correlates with the concentration of the trigger and facilitates detection of the trigger concentration.

E428. The method of embodiment 426 or 427, wherein each fluorescent dye is different from one another.

E429. The method of embodiment 428, wherein each fluorescent dye exhibits distinct excitation and emission spectra from one another.

E430. The method of any one of embodiments 426-429, wherein each fluorescent dye is selected from 7-nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUETM dye), 5-(dimethylamino)naphthalene-1 -sulfonyl (Dansyl), pyrene, 7-amino-3- {[(2,5-dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350TM), 6,8-difluoro-7-hydroxy-4-methylcoumarin (MARINA BLUETM dye), N-(2-aminoethyl)- 4-{5-[4-(dimethylamino)phenyl]-1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYLTM dye), 2, 3,5,6- Tetramethyl-1 H,7H-pyrazolo[1 ,2-a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4- (ethylamino)naphthalen-2-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUETM dye), tris(N,N-diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrolidin-1 -yl)oxy]carbonyl}piperidin- 1 -yl)-2-oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405TM), N,N-diethylethanaminium [9-{6- [(2,5-dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H- benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430TM), 1 -[({4-[(7-nitro-2,1 ,3- benzoxadiazol-4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSYTM dye), fluorescein, 2-(6- amino-3-iminio-4,5-disulfonato-3H-xanthen-9-yl)-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488TM), ’',”-Difluoro-”,”-dihydroxy-3H-spiro[isobenzofuran-1 ,”-xanthen]-3-one (OREGON GREENTM 488), 1 ,3,5,7, 8-pentamethyl-4,4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPYTM 493/503), rhodamine green, 13-[2-carboxy-3,4,6-trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 - yl)oxy-6-oxohexyl]amino]-2-oxoethyl]sulfanylphenyl]-7,7,9,17,19,19-hexamethyl-2-oxa-6,20- diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa-1 (14),3,5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546TM), 2-[5-[3,3-dimethyl-5-sulfo-1 -(3-sulfopropyl)indol-1 -ium-2-yl]penta-2,4- dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5- sulfonic acid (ALEXA FLUOR 647TM), and rhodamine red;

E431 . The method of any one of embodiments 416-430, wherein steps (a’) through (b’) have been performed on a plurality of compounds, thereby screening a compound library to identify a compound that inhibits or promotes LLPS of the target macromolecule in the cell.

E432. The method of embodiment 431 , wherein the screening is high-throughput screening.

E433. The method of embodiment 431 or 432, wherein the compound library comprises from 10 to 100,000 compounds, or more.

E434. The method of any one of embodiments 416-433, wherein the cell is a eukaryotic cell.

E435. The method of embodiment 434, wherein the eukaryotic cell is a mammalian cell.

E436. The method of embodiment 435, wherein the mammalian cell is a human cell.

E437. The method of any one of embodiments 416-436, wherein the cell naturally expresses the target macromolecule and undergoes LLPS or the cell is induced to express the target macromolecule.

E438. The method of any one of embodiments 416-437, wherein:

(a) the cell comprises a non-target macromolecule;

(b) the target macromolecule is a protein or nucleic acid; and/or (c) optionally wherein the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that phase separation of a condensate system that comprises the target macromolecule.

E439. The method of any one of embodiments 417-438, wherein the trigger induces oxidative stress in the cell, thereby promoting localization of the target macromolecule to cytoplasmic stress granules.

E440. The method of embodiment 439, wherein the trigger induces oxidative stress is sodium arsenite.

E441 . The method of any one of embodiments 417-440, wherein the trigger induces a change in a chemical modification state of the target macromolecule, thereby causing the target macromolecule to localize to a condensate in the cell.

E442. The method of embodiment 441 , wherein the chemical modification is a post-translation modification, such as phosphorylation, methylation, or acetylation.

E443. The method of embodiment 442, wherein the chemical modification is methylation, and wherein the trigger induces a change in methylation state is adenosine dialdehyde.

E444. The method of any one of embodiments 417-443, wherein the trigger induces formation of nucleolar caps in the cell, thereby promoting localization of the target macromolecule to a nucleolar cap.

E445. The method of embodiment 444, wherein the trigger induces formation of nucleolar caps in the cell is actinomycin D.

E446. The method of any one of embodiments 416-445, wherein step (b’) is performed by way of fluorescent microscopy.

E447. The method of any one of embodiments 416-446, wherein the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4-ALK, FUS-CHOP, YAP, TAZ or MBNL1 .

E448. A method of determining whether a compound selectively inhibits or promotes LLPS of a target macromolecule, the method comprising:

(a’) introducing, into a cell, the compound identified as one that selectively promotes or inhibits LLPS of the target macromolecule in vitro, wherein the cell comprises the target macromolecule and the non-target macromolecule; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-target), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, and

E449. The method of embodiment 448, wherein the reference compound is a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule.

E450. The method of embodiment 448 or 449, wherein a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule in a cell, is added to the cell.

E451 . The method of embodiment 449 or 450, wherein the trigger of LLPS is a protein, nucleic acid, salt, or polyethylene glycol (PEG), optionally wherein the PEG has an average molecular weight of from 600 Da to 20 kDa, optionally wherein the PEG has an average molecular weight of about 10 kDa.

E452. The method of any one of embodiments 448-451 , wherein the target macromolecule is a protein or nucleic acid.

E453. The method of embodiment 452, wherein the nucleic acid is DNA or RNA.

E454. The method of embodiment 453, wherein the RNA is mRNA, hnRNA, non-coding RNA, rRNA, tRNA, IncRNSA, or miRNA.

E455. The method of any one of embodiments 448-454, wherein the non-target macromolecule is a protein or a nucleic acid. E456. The method of embodiment 455, wherein the nucleic acid is an RNA transcript, a specific RNA, a DMPK transcript, an AR transcript, total RNA, poly adenylated RNA or m6A marked RNA.

E457. The method of any one of embodiments 448-456, wherein the compound is added to the cell as a stock mixture comprising the compound.

E458. The method of any one of embodiments 449-457, wherein the trigger is added to the cell as a portion of a stock mixture comprising the trigger.

E459. The method of embodiment 457 or 458, wherein:

E460. The method of embodiment 459, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FDcompound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDcompound) correlates with the concentration of the compound and facilitates detection of the compound concentration; and the stock mixture comprising the trigger further comprises a fluorescent dye (FDtrigger) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDtrigger) correlates with the concentration of the trigger and facilitates detection of the trigger concentration.

E461 . The method of embodiment 459 or 460, wherein each fluorescent dye is different from one another.

E462. The method of embodiment 461 , wherein each fluorescent dye exhibits distinct excitation and emission spectra from one another.

E463. The method of any one of embodiments 459-462, wherein each fluorescent dye is selected from 7-nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUETM dye), 5-(dimethylamino)naphthalene-1 -sulfonyl (Dansyl), pyrene, 7-amino-3- {[(2,5-dioxopyrrolidin-1 -yl)oxy]-2-oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350TM), 6,8-difluoro-7-hydroxy-4-methylcoumarin (MARINA BLUETM dye), N-(2-aminoethyl)- 4-{5-[4-(dimethylamino)phenyl]-1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYLTM dye), 2, 3,5,6- Tetramethyl-1 H,7H-pyrazolo[1 ,2-a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4- (ethylamino)naphthalen-2-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUETM dye), tris(N,N-diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrolidin-1 -yl)oxy]carbonyl}piperidin- 1 -yl)-2-oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405TM), N,N-diethylethanaminium [9-{6- [(2,5-dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H- benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430TM), 1 -[({4-[(7-nitro-2,1 ,3- benzoxadiazol-4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSYTM dye), fluorescein, 2-(6- amino-3-iminio-4,5-disulfonato-3H-xanthen-9-yl)-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488TM), 2',7'-Difluoro-3',6'-dihydroxy-3H-spiro[isobenzofuran-1 ,9'-xanthen]-3-one (OREGON GREENTM 488), 1 ,3,5,7, 8-pentamethyl-4,4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPYTM 493/503), rhodamine green, 13-[2-carboxy-3,4,6-trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 - yl)oxy-6-oxohexyl]amino]-2-oxoethyl]sulfanylphenyl]-7,7,9,17,19,19-hexamethyl-2-oxa-6,20- diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa-1 (14),3,5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546TM), 2-[5-[3,3-dimethyl-5-sulfo-1 -(3-sulfopropyl)indol-1 -ium-2-yl]penta-2,4- dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5- sulfonic acid (ALEXA FLUOR 647TM), and rhodamine red;

E464. The method of any one of embodiments 448-463, wherein method identifies the compound that selectively promotes or inhibits homotypic LLPS of the target molecule.

E465. The method of any one of embodiments 448-464, wherein the method identified the compound that selectively promotes or inhibits heterotypic LLPS of the target macromolecule and the non-target macromolecule.

E466. The method of any one of embodiments 448-465, wherein steps (a’) through (b’) have been performed on a plurality of compounds, thereby screening a compound library to identify a compound that inhibits or promotes LLPS of the target macromolecule in the cell.

E467. The method of embodiment 466, wherein the screening is high-throughput screening.

E468. The method of embodiment 466 or 467, wherein the compound library comprises from 10 to 100,000 compounds, or more.

E469. The method of any one of embodiments 448-468, wherein the microdroplet comprises a cell lysate or cells.

E470. The method of embodiment 469, wherein the cells are from a diseased cell line.

E471 . The method of any one of embodiments 448-470, wherein the cell is a eukaryotic cell.

E472. The method of embodiment 471 , wherein the eukaryotic cell is a mammalian cell.

E473. The method of embodiment 472, wherein the mammalian cell is a human cell. E474. The method of any one of embodiments 448-473, wherein the cell naturally expresses the target macromolecule and undergoes LLPS.

E475. The method of any one of embodiments 448-474, wherein the cell is induced to express the target macromolecule.

E476. The method of any one of embodiments 449-475, wherein the trigger induces oxidative stress in the cell, thereby promoting localization of the target macromolecule to cytoplasmic stress granules.

E477. The method of embodiment 476, wherein the trigger that induces oxidative stress is sodium arsenite.

E478. The method of any one of embodiments 449-477, wherein the trigger induces a change in a chemical modification state of the target macromolecule, thereby causing the target macromolecule to localize to a condensate in the cell.

E479. The method of embodiment 478, wherein the chemical modification is a post-transcriptional modification, such as phosphorylation, methylation, or acetylation.

E480. The method of embodiment 479, wherein the trigger induces a change in methylation state is adenosine dialdehyde.

E481 . The method of any one of embodiments 449-480, wherein the trigger induces formation of nucleolar caps in the cell, thereby promoting localization of the target macromolecule to a nucleolar cap.

E482. The method of embodiment 481 , wherein the trigger induces formation of nucleolar caps in the cell is actinomycin D.

E483. The method of any one of embodiments 448-482, wherein step (b’) is performed by way of fluorescent microscopy.

E484. The method of any one of embodiments 448-483, wherein the compound is identified as one that binds to, and/or modulates the activity of the target macromolecule.

E485. The method of any one of embodiments 448-484, wherein:

E486. The method of any one of embodiments 448-485, wherein the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4-ALK, FUS-CHOP, YAP, TAZ or MBNL1 .

E487. A method of identifying a compound useful for treating a disease in an individual in need thereof, the method comprising performing the method of any one of embodiments 1 -236, wherein the phase transition of the condensate is associated with the disease, and wherein the compound is identified as one that modulates one or more phase transition characteristics of the condensate, thereby identifying a compound useful for treating the disease.

E488. A method of identifying a compound useful for treating a disease in an individual in need thereof, the method comprising performing the method of any one of embodiments 237-486, wherein the LLPS of the target macromolecule is associated with the disease, and wherein the compound is identified as one that inhibits the LLPS of the target macromolecule, thereby identifying a compound useful for treating the disease.

E489. The method of embodiment 487 or 488, the method further comprising administering a therapeutically effective amount of the compound to an individual diagnosed as having the disease.

E490. A method of identifying a target macromolecule for use in the method of any one of embodiments 1 -486, the method comprising:

(a) in silico screening a multimodal data set from a plurality of human biological samples to identify a plurality of genetic variations that distinguish a human disease state from a human healthy state;

(b) in silico screening a multimodal data set from a plurality of in vitro disease relevant cell line models to identify a plurality of genetic variations that distinguish a disease state from a healthy state;

(c) analyzing the plurality of genetic variations to identify one or a subset of genetic variations that lead to a change in concentration of the target macromolecule, a chemical alternation of the target macromolecule, and/or a change in the endogenous environment of the target macromolecule;

(a’) determining that the change in concentration, the chemical alteration and/or change in endogenous environment of the target macromolecule leads to aberrant condensation behavior in the disease-associated model; and

(b’) determining that the change described in (a’) does not lead to an aberrant condensation behavior in biological samples from healthy volunteers or non-diseased cell models. E491 . The method of embodiment 490, wherein the target macromolecule is a protein capable of undergoing phase transition or LLPS, or localizing into biomolecular condensates.

E492. The method of embodiment 490, wherein the target macromolecule is a protein that can undergo LLPS or localize into bimolecular condensates as a result of pathogenic mutation.

E493. The method of embodiment 490, wherein the multimodal data modalities comprise one or many of the following: DNA sequencing data describing genetic variations, transcriptomic data, proteomic data, dependency data acquired by measuring cell viability upon knockdown or knockout of the target gene of interest.

E494. The method of embodiment 490, wherein the chemical alternation of the target macromolecule comprises one or more genetic variations in the protein sequence.

E495. The method of embodiment 494, wherein the one or more genetic variations in the protein sequence is missense mutations, deletions, fusions, truncations, and/or frameshift mutations.

E496. The method of embodiment 495, wherein the chemical alternation of the target macromolecule comprises post-translational modifications.

E497. The method of embodiment 496, wherein the post-translational modification is phosphorylation, acetylation, sumoylation, ubiquitination, myristoylation, and/or palmitoylation.

E498. The method of or any of the embodiments 492, wherein one or more of the mutations are enriched in one or more regions of a protein that are associated with the candidate target protein.

E499. The method of embodiment 498, wherein the one or more regions of a protein are a low complexity region, such as a disordered region of the protein.

E500. The method of embodiment 498, wherein one or more regions of a protein carry or support a functional role, such as a catalytic domain or an interaction interface with another molecule, or a region that is involved in allosterically regulating the function of a catalytic domain or an interaction interface.

E501 . The method of any one of embodiments 498-500, wherein the one or more regions of a protein that are associated with LLPS or phase transition is based on experimentally obtained wet lab data.

E502. The method of any one of embodiments 498-501 , wherein the one or more regions of a protein that are associated with LLPS or phase transition is based on statistical analysis of the enrichment of protein domains into biomolecular condensate systems using the composition of previously characterized condensate systems as the input.

E503. The method of any one of embodiments 498-502, wherein the one or more regions of a protein that are associated with LLPS or phase transition is determined by using predictive models that link a protein sequence and its altered form to the condensation propensity.

E504. The method of any one of embodiments 598-503, wherein the one or more regions of a protein that are associated with LLPS or phase transition is determined via a saturation mutagenesis analysis across the sequence that identifies regions of the protein wherein condensation behavior is sensitive to alterations in the sequence.

E505. The method of any one of embodiments 490-504, wherein the change in concentration as a result of the genetic variations lead to an altered condensation state of the protein as can be deduced from a comparison to the endogenous concentration of the target molecule and its saturation concentration (csat) of the target macromolecule.

E506. The method of any one of embodiments 490-505, wherein the genetic variations cause the concentration of the macromolecule to increase above the endogenous saturation concentration of the target molecule.

E507. The method of any one of embodiments 490-506, wherein the genetic variations are a fusion of two genes, a missense or truncation mutation

E508. The method of any one of embodiments 490-507, wherein the genetic variation results in reduced degradation of the target macromolecule.

E509. The method of any one of embodiments 490-508, wherein the genetic variations cause the concentration of the macromolecule to reduce below the endogenous saturation concentration.

E510. The method of any one of embodiments 490-509, wherein the genetic variations are a deletion mutation, a missense, or truncations mutation

E511 . The method of any one of embodiments 490-510, wherein the genetic variation results in reduced rate of protein production.

E512. The method of any one of embodiments 490-511 , wherein the change in the endogenous environment of the target macromolecule comprises a change in the availability of one or more endogenous modulators of LLPS or phase transition. E513. The method of any one of embodiments 490-512, wherein the change in the endogenous environment of the target macromolecule comprises a change in the level of molecular crowding.

E514. The method of any one of embodiments 490-513, wherein the change in the endogenous environment comprises a reduction in the concentration of the key effectors of LLPS or phase transition.

E515. The method of any one of embodiments 490-514, wherein the key effectors of LLPS or phase transition are scaffolding proteins or RNA.

Other Embodiments

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations following, in general, the principles and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

Claims

1 . A method for screening a plurality of compounds to identify condensate modulators that inhibit or promote liquid-liquid phase separation (LLPS) of a target macromolecule, the method comprising:

(b) determining, within each of the microdroplets, whether the target macromolecule has undergone LLPS, thereby identifying the concentration of the compound at which the target macromolecule undergoes LLPS; and

2. A method of determining whether a compound inhibits or promotes LLPS of a target macromolecule, the method comprising:

(a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, and wherein the concentration of the compound varies among the plurality of microdroplets;

(c) determining whether the compound promotes or inhibits LLPS, wherein: the determining is by comparing the concentration of the compound at which the target macromolecule undergoes LLPS as compared to a concentration of a reference compound at which the target macromolecule undergoes LLPS, the compound promotes LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is less than the concentration of the reference compound at which the target macromolecule undergoes LLPS, and the compound inhibits LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is greater than the concentration of the reference compound at which the target macromolecule undergoes LLPS, (a’) introducing, into a cell, the compound identified in (c) as one that promotes or inhibits LLPS of the target macromolecule in vitro, wherein the cell comprises the target macromolecule; and

3. A method of determining whether a compound selectively inhibits or promotes LLPS of a target macromolecule, the method comprising:

(a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, a non-target macromolecule and wherein the concentration of the compound varies among the plurality of microdroplets; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-tar et) , wherein fluorescence of each non-target fluorescent dye (FDnon-tar et) facilitates detection of one non-target macromolecule,

(c) determining whether the compound selectively promotes or inhibits LLPS of the target macromolecule, wherein: the determining is by comparing the concentration of the compound at which the target macromolecule undergoes LLPS as compared to a concentration of at which the non-target macromolecule undergoes LLPS, the compound promotes LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is less than the concentration of the compound at which the non- target macromolecule undergoes LLPS, and the compound inhibits LLPS when the concentration of the compound at which the target macromolecule undergoes LLPS is greater than the concentration of the compound at which the nontarget macromolecule undergoes LLPS.

4. A method of identifying condensate modulators, the method comprising contacting a condensate modulator with a composition comprising a target macromolecule and measuring one or more phase transition characteristics of the condensate.

5. The method of claim 4, wherein the method comprising:

6. A method of identifying selective condensate modulators, the method comprising contacting a condensate modulator with a composition comprising a target macromolecule and a non-target macromolecule, and measuring one or more phase transition characteristics of the condensate.

7. The method of claim 6, the method comprising:

(a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, a non-target macromolecule and wherein the concentration of the compound varies among the plurality of microdroplets; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non- covalently bound to a fluorescent dye (FDnon-target), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule;

(b) determining, within each of the microdroplets, whether the target macromolecule has undergone phase transition, and whether the non-target macromolecule has undergone phase transition, thereby identifying the concentration of the compound at which the one or more phase transition characteristics change and the concentration of the compound at which the non-target macromolecule undergoes phase transition; (c) determining whether the compound selectively modulates one or more phase transition characteristics of the condensate, wherein: the determining is by comparing the concentration of the compound at which the one or more phase transition characteristics change as compared to a concentration of at which the non-target macromolecule undergoes phase transition.

8. A method of identifying selective condensate modulators, the method comprising contacting a condensate modulator with a composition comprising a target macromolecule and a non-target macromolecule, and measuring one or more phase transition characteristics of the condensate.

9. The method of claim 8, the method comprising:

(a) producing a plurality of microdroplets in vitro, wherein each of the microdroplets comprises the compound, the target macromolecule, a non-target macromolecule and wherein the concentration of the compound varies among the plurality of microdroplets; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non- covalently bound to a fluorescent dye (FDnon-target), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule,

(c) determining whether the compound selectively modulates one or more phase transition characteristics of the condensate, wherein: the determining is by comparing the concentration of the compound at which the one or more phase transition characteristics change as compared to a concentration of at which the non-target macromolecule undergoes phase transition,

(a’) introducing, into a cell, the compound identified in (c) as one that selectively modulates one or more phase transition characteristics of the condensate, in vitro, wherein the cell comprises the target macromolecule and the non-target macromolecule; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non- covalently bound to a fluorescent dye (FDnon-target), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, and (b’) assessing whether the compound selectively modulates one or more phase transition characteristics of the condensate in the cell, wherein: the determining is by comparing the concentration of the compound at which the one or more phase transition characteristics change in the cell, as compared to a concentration of the compound at which the non-target macromolecule undergoes phase transition in the cell.

10. A method of determining whether a compound selectively inhibits or promotes LLPS of a target macromolecule, the method comprising:

(a’) introducing, into a cell, the compound identified in (c) as one that selectively promotes or inhibits LLPS of the target macromolecule in vitro, wherein the cell comprises the target macromolecule and the non-target macromolecule; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtar et) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-target) , wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, and

11. A method of determining whether a compound inhibits or promotes LLPS of a target macromolecule in a cell, the method comprising:

12. A method of determining whether a compound selectively inhibits or promotes LLPS of a target macromolecule, the method comprising:

(a’) introducing, into a cell, the compound identified as one that selectively promotes or inhibits LLPS of the target macromolecule in vitro, wherein the cell comprises the target macromolecule and the non-target macromolecule; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDnon-target) , wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, and

13. A method of identifying a compound that modulates one or more phase characteristics of a condensate in a cell, the method comprising:

(a’) introducing, into a cell, the compound identified as one that modulates one or more phase transition characteristics of the condensate in vitro, wherein the cell comprises the target macromolecule; and

14. A method of identifying selective condensate modulators, the method comprising contacting a condensate modulator with a composition comprising a target macromolecule and a non-target macromolecule, and measuring one or more phase transition characteristics of the condensate in a cell.

15. The method of claim 14, the method comprising:

(a’) introducing, into a cell, the compound identified as one that selectively modulates one or more phase transition characteristics of the condensate, in vitro, wherein the cell comprises the target macromolecule and the non-target macromolecule; and each of the target macromolecule is, independently, covalently or non-covalently bound to a fluorescent dye (FDtarget), wherein fluorescence of each target fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and each of the non-target macromolecule is, independently, covalently or non- covalently bound to a fluorescent dye (FDnon-target), wherein fluorescence of each non-target fluorescent dye (FDnon-target) facilitates detection of one non-target macromolecule, and

16. The method of claim 4, the method further comprising:

17. The method of any one of claims 1 , 2, 5, 11 , 13 and 16, wherein the reference compound is a trigger of LLPS or phase transition that is known to promote or inhibit LLPS or phase transition of the target macromolecule.

18. The method of any one of claims 3, 6-10, 12, 14-15, and 17, wherein the non-target macromolecule is a protein or a nucleic acid.

19. The method of claim 17, wherein the non-target macromolecule is a specific RNA, a DMPK transcript or AR transcript, total RNA, poly adenylated RNA, RNA transcript, or m6A marked RNA.

20. The method of any one of claims 1 -3, 5, 7, 9-10, and 16-19, wherein the method comprises the step of producing the microdroplets on a microfluidic chip.

21 . The method of any one of claims 1 -3, 5, 7, 9, 10, and 16-20, wherein the microdroplets comprise a trigger of LLPS that is known to promote or inhibit LLPS of the target macromolecule in vitro.

22. The method of any one of claims 1 -3, 5, 7, 9, 10, and 16-21 , wherein the microdroplet comprises a cell lysate or a cell.

23. The method of any one of claims 1 -3, 5, 7, 9, 10, and 16-22, wherein each microdroplet comprises a portion of a stock mixture comprising the target macromolecule and a portion of a stock mixture comprising the compound.

24. The method of any one of claims 1 -3, 5, 7, 9, 10, and 16-23, wherein each microdroplet further comprises a portion of a stock mixture comprising the trigger.

25. The method of claim 23 or 24, wherein each microdroplet is produced by automated mixing of the stock mixtures.

26. The method of any one of claims 1 -3, 5, 7, 9, 10, and 16-25, wherein the step of determining, within each of the microdroplets, the target macromolecule is visualized by fluorescent microscopy to determine if the target macromolecule has undergone LLPS.

27. The method of any one of claims 1 -3, 5, 7, 9, 10, and 16-26, wherein a finding that the target macromolecule is present within the microdroplet in discrete puncta is taken as an indication that the target macromolecule has undergone LLPS, and a finding that the target macromolecule is present in a homogenous distribution within the microdroplet, absent discrete puncta, is taken as an indication that the target macromolecule has not undergone LLPS.

28. The method of any one of claims 1 -3, 5, 7, 9, 10, and 16-27, wherein the concentration of the compound at which the target macromolecule undergoes LLPS is normalized relative to a positive reference microdroplet and/or a negative reference microdroplet, wherein the positive reference microdroplet comprises the target macromolecule and a trigger of LLPS, and the negative reference microdroplet comprises the target macromolecule.

29. The method of any one of claims 1 -3, 5, 7, 9, 10, and 16-28, wherein the microdroplet comprises a cell from a diseased cell line.

30. The method of any one of claims 1 -3, 5, 7, 9, 10, and 16-29, wherein each microdroplet comprises a portion of a stock mixture comprising the target macromolecule, a portion of a stock mixture comprising the compound and a position of a stock mixture comprising a non-target macromolecule.

31 . The method of any one of claims 1 -3, 5, 7, 9, 10, and 16-30, wherein the microdroplet comprises a non-target macromolecule; optionally wherein the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that modulates phase transition characteristics of a condensate system that comprises the target macromolecule.

32. The method of any one of claims 1 -3, 5, 7, 9, 10, and 16-31 , wherein steps (a) through (c) are performed on a plurality of compounds, thereby screening a compound library to identify a compound that inhibits or promotes LLPS of the target macromolecule in vitro.

33. The method of claim 32, wherein the screening is high-throughput screening.

34. The method of claim 32 or 33, wherein the compound library comprises from 10 to 100,000 compounds, or more.

35. The method of any one of claims 2, 9-13, and 15-18, wherein the compound is added to the cell as a stock mixture comprising the compound.

36. The method of any one of claims 2, 9-13, 15-18, and 35, wherein a trigger of phase transition that is known to modulate one or more phase transition characteristics of a condensate in a cell, is added to the cell.

37. The method of claim 35 or 36, wherein the trigger is added to the cell as a portion of a stock mixture comprising the trigger.

38. The method of any one of claims 2, 9-13, 15-18, and 35-37, wherein steps (a’) through (b’) have been performed on a plurality of compounds, thereby screening a compound library to identify a compound that inhibits or promotes LLPS of the target macromolecule in the cell.

39. The method of any one of claims 2, 9-13, 15-18, and 35-38, wherein the cell is a eukaryotic cell.

40. The method of claim 39, wherein the eukaryotic cell is a mammalian cell.

41 . The method of claim 40, wherein the mammalian cell is a human cell.

42. The method of any one of claims 2, 9-13, 15-18, and 35-41 , wherein the cell naturally expresses the target macromolecule and undergoes LLPS.

43. The method of any one of claims 2, 9-13, 15-18, and 35-42, wherein the cell is induced to express the target macromolecule.

44. The method of any one of claims 2, 9-13, 15-18, and 35-43, wherein the cell is from a diseased cell line.

45. The method of any one of claims 2, 9-13, 15-18, and 35-44, wherein step (b’) is performed by way of fluorescent microscopy.

46. The method of any one of claims 2, 9-13, 15-18, and 35-45, wherein:

(a) the cell comprises a non-target macromolecule;

(b) the target macromolecule is a protein or nucleic acid; and/or

(c) optionally wherein the compound is not identified as one that binds to, or modulates the activity of, the target macromolecule, and the compound is not identified as one that modulates binding of the target macromolecule to the non-target macromolecule, but wherein the compound is identified as one that phase separation of a condensate system that comprises the target macromolecule.

47. The method of any one of claims 23-46, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FD_COm_Pound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FD_COm_Pound) correlates with the concentration of the compound and facilitates detection of the compound concentration.

48. The method of any one of claims 23-46, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FD_COm_Pound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FD_COm_Pound) correlates with the concentration of the compound and facilitates detection of the compound concentration; and each of the target macromolecule is, independently, covalently or noncovalently bound to a fluorescent dye (FDtarget), whereby fluorescence of one fluorescent dye (FDtarget) facilitates detection of one target macromolecule.

49. The method of any one of claim 23-46, wherein: the stock mixture comprising the compound further comprises a fluorescent dye (FD_COm_Pound) at a fixed concentration, whereby fluorescence of the fluorescent dye (FD_COm_Pound) correlates with the concentration of the compound and facilitates detection of the compound concentration; each of the target macromolecule is, independently, covalently, or noncovalently bound to a fluorescent dye (FDtarget), whereby fluorescence of each fluorescent dye (FDtarget) facilitates detection of one target macromolecule, and the stock mixture comprising the trigger further comprises a fluorescent dye (FDtdgger) at a fixed concentration, whereby fluorescence of the fluorescent dye (FDtrigger) correlates with the concentration of the trigger and facilitates detection of the trigger concentration.

50. The method of any one of claims 47-49, wherein each fluorescent dye is different from one another.

51 . The method of any one of claims 47-50, wherein each fluorescent dye exhibits distinct excitation and emission spectra from one another.

52. The method of any one of claims 47-51 , wherein each fluorescent dye is selected from 7- nitrobenz-2-oxa-1 ,3-diazol-4-yl (NBD), methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin acetate (AMCA), 6,8-difluoro-7-hydroxy-3-carboxycoumarin (PACIFIC BLUE™ dye), 5-(dimethylamino)naphthalene-1 -sulfonyl (Dansyl), pyrene, 7-amino-3-{[(2,5-dioxopyrrolidin-1 - yl)oxy]-2-oxoethyl}-4-methyl-2-oxo-2H-chromene-6-sulfonic acid (ALEXA FLUOR 350™), 6,8-difluoro- 7-hydroxy-4-methylcoumarin (MARINA BLUE™ dye), N-(2-aminoethyl)-4-{5-[4- (dimethylamino)phenyl]-1 ,3-oxazol-2-yl}benzenesulfonamide (DAPOXYL™ dye), 2,3,5,6-Tetramethyl- 1 H,7H-pyrazolo[1 ,2-a]pyrazole-1 ,7-dione (Bimane), 4-{[4-(Diethylamino)phenyl][4- (ethylamino)naphthalen-2-yl]methylidene}-N,N-diethylcyclohexa-2,5-dien-1 -iminium (CASCADE BLUE™ dye), tris(N,N-diethylethanaminium) 8-[2-(4-{[(2,5-dioxopyrrol idin-1 -yl)oxy]carbonyl}piperidin- 1 -yl)-2-oxoethoxy]pyrene-1 ,3,6-trisulfonate (ALEXA FLUOR 405™), N,N-diethylethanaminium [9-{6- [(2,5-dioxopyrrolidin-1 -yl)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H- benzo[g]chromen-6-yl]methanesulfonate (ALEXA FLUOR 430™), 1 -[({4-[(7-nitro-2,1 ,3-benzoxadiazol- 4-yl)amino]phenyl}acetyl)oxy]pyrrolidine-2, 5-dione (QSY™ dye), fluorescein, 2-(6-amino-3-iminio-4,5- disulfonato-3H-xanthen-9-yl)-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)benzoate (ALEXA FLUOR 488™), 2',7'-Difluoro-3',6'-dihydroxy-3H-spiro[isobenzofuran-1 ,9'-xanthen]-3-one (OREGON GREEN™ 488), 1 ,3,5,7,8-pentamethyl-4,4-difluorro-4-bora-3a,4a-diaza-s-indacene (BODIPY™ 493/503), rhodamine green, 13-[2-carboxy-3,4,6-trichloro-5-[2-[[6-(2,5-dioxopyrrolidin-1 -yl)oxy-6- oxohexyl]amino]-2-oxoethyl]sulfanylphenyl]-7,7,9,17,19,19-hexamethyl-2-oxa-6,20- diazapentacyclo[12.8.0.03,12.05,10.016,21 ]docosa-1 (14),3,5,10,12,15,21 -heptaene-4,22-disulfonate (ALEXA FLUOR 546™), 2-[5-[3,3-dimethyl-5-sulfo-1 -(3-sulfopropyl)indol-1 -ium-2-yl]penta-2,4- dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-1 -(3-sulfopropyl)indole-5- sulfonic acid (ALEXA FLUOR 647™), and rhodamine red; and/or wherein one or more of the fluorescent dye is a fluorescent protein selected from GFP, mNeonGreen, mRFP, mCherry, and mPlum.

53. The method of any one of claims 16-52, wherein the trigger of LLPS is a protein, nucleic acid, salt, or polyethylene glycol (PEG).

54. The method of claim 53, wherein the polyethylene glycol (PEG) has an average molecular weight of from 600 Da to 20 kDa or about 10 kDa.

55. The method of any one of claims 16-54, wherein the trigger induces oxidative stress in the cell, thereby promoting localization of the target macromolecule to cytoplasmic stress granules.

56. The method of claim 16-52, wherein the trigger is sodium arsenite.

57. The method of any one of claims 16-52, wherein the trigger induces a change in a chemical modification state of the target macromolecule, thereby causing the target macromolecule to localize to a condensate in the cell.

58. The method of claim 57, wherein the chemical modification is a post-translation modification, such as phosphorylation, methylation, or acetylation.

59. The method of claim 16-52, wherein the trigger is adenosine dialdehyde.

60. The method of any one of claims 16-52, wherein the trigger induces formation of nucleolar caps in the cell, thereby promoting localization of the target macromolecule to a nucleolar cap.

61 . The method of claim 16-52, wherein the trigger is actinomycin D.

62. The method of any one of claims 1 -60, wherein the target macromolecule is a protein or nucleic acid.

63. The method of claim 62, wherein the nucleic acid is DNA or RNA.

64. The method of claim 63, wherein the RNA is mRNA, hnRNA, non-coding RNA, rRNA, tRNA,

IncRNSA, or miRNA.

65. The method of any one of claims 1 -62, wherein the target macromolecule is FUS, G3BP1 , DDX3X, Androgen receptor (AR), YTHDC1 , EML4-ALK, FUS-CHOP, YAP, TAZ or MBNL1 .

66. The method of any one of claims 1 -65, wherein the compound is identified as one that binds to and/or modulates the activity of the target macromolecule.

67. The method of any one of claims 1 -66, wherein method identifies the compound that selectively promotes or inhibits homotypic LLPS of the target molecule.

68. The method of any one of claims 3, 6-10, 12, and 14-67, wherein the method identified the compound that selectively promotes or inhibits heterotypic LLPS of the target macromolecule and the non-target macromolecule.

69. The method of any one of claims 3, 6-10, 12, and 14-67, wherein:

70. A method of identifying a compound useful for treating a disease in an individual in need thereof, the method comprising performing the method of any one of claims 4-9, and 13-69, wherein the phase transition of the condensate is associated with the disease, and wherein the compound is identified as one that modulates one or more phase transition characteristics of the condensate, thereby identifying a compound useful for treating the disease.

71 . A method of identifying a compound useful for treating a disease in an individual in need thereof, the method comprising performing the method of any one of claims 1 -3, 10-12, and 16-70, wherein the LLPS of the target macromolecule is associated with the disease, and wherein the compound is identified as one that inhibits the LLPS of the target macromolecule, thereby identifying a compound useful for treating the disease.

72. The method of claim 70 or 71 , the method further comprising administering a therapeutically effective amount of the compound to an individual diagnosed as having the disease.

73. A method of identifying a target macromolecule for use in the method of any one of claims 1 - 72, the method comprising:

(a) in silico screening a multimodal data set from a plurality of human biological samples to identify a plurality of genetic variations that distinguish a human disease state from a human healthy state; (b) in silico screening a multimodal data set from a plurality of in vitro disease relevant cell line models to identify a plurality of genetic variations that distinguish a disease state from a healthy state;

(b’) determining that the change described in (a’) does not lead to an aberrant condensation behavior in biological samples from healthy volunteers or non-diseased cell models.

74. The method of claim 73, wherein the target macromolecule is a protein capable of undergoing phase transition or LLPS, or localizing into biomolecular condensates.

75. The method of claim 73, wherein the target macromolecule is a protein that can undergo phase transition or LLPS, or localize into bimolecular condensates as a result of pathogenic mutation.

76. The method of claim 73, wherein the multimodal data modalities comprise one or many of the following: DNA sequencing data describing genetic variations, transcriptomic data, proteomic data, dependency data acquired by measuring cell viability upon knockdown or knockout of the target gene of interest.

77. The method of claim 73, wherein the chemical alternation of the target macromolecule comprises one or more genetic variations in the protein sequence.

78. The method of claim 77, wherein the one or more genetic variations in the protein sequence is missense mutations, deletions, fusions, truncations, and/or frameshift mutations.

79. The method of claim 78, wherein the chemical alternation of the target macromolecule comprises post-translational modifications.

80. The method of claim 79, wherein the post-translational modification is phosphorylation, acetylation, sumoylation, ubiquitination, myristoylation, and/or palmitoylation.

81 . The method of or any of the claims 75, wherein one or more of the mutations are enriched in one or more regions of a protein that are associated with the candidate target protein.

82. The method of claim 81 , wherein the one or more regions of a protein are a low complexity region, such as a disordered region of the protein.

83. The method of claim 81 , wherein one or more regions of a protein carry or support a functional role, such as a catalytic domain or an interaction interface with another molecule, or a region that is involved in allosterically regulating the function of a catalytic domain or an interaction interface.

84. The method of any one of claims 81 -83, wherein the one or more regions of a protein that are associated with LLPS or phase transition is based on experimentally obtained wet lab data.

85. The method of any one of claims 81 -84, wherein the one or more regions of a protein that are associated with LLPS or phase transition is based on statistical analysis of the enrichment of protein domains into biomolecular condensate systems using the composition of previously characterized condensate systems as the input.

86. The method of any one of claims 81 -85, wherein the one or more regions of a protein that are associated with LLPS or phase transition is determined by using predictive models that link a protein sequence and its altered form to the condensation propensity.

87. The method of any one of claims 81 -86, wherein the one or more regions of a protein that are associated with LLPS or phase transition is determined via a saturation mutagenesis analysis across the sequence that identifies regions of the protein wherein condensation behavior is sensitive to alterations in the sequence.

88. The method of any one of claims 73-87, wherein the change in concentration as a result of the genetic variations lead to an altered condensation state of the protein as can be deduced from a comparison to the endogenous concentration of the target molecule and its saturation concentration (csat) of the target macromolecule.

89. The method of any one of claims 73-88, wherein the genetic variations cause the concentration of the macromolecule to increase above the endogenous saturation concentration of the target molecule.

90. The method of any one of claims 73-89, wherein the genetic variations are a fusion of two genes, a missense or truncation mutation

91 . The method of any one of claims 73-90, wherein the genetic variation results in reduced degradation of the target macromolecule.

92. The method of any one of claims 73-91 , wherein the genetic variations cause the concentration of the macromolecule to reduce below the endogenous saturation concentration.

93. The method of any one of claims 73-92, wherein the genetic variations are a deletion mutation, a missense, or truncations mutation

94. The method of any one of claims 73-93, wherein the genetic variation results in reduced rate of protein production.

95. The method of any one of claims 73-94, wherein the change in the endogenous environment of the target macromolecule comprises a change in the availability of one or more endogenous modulators of LLPS or phase transition.

96. The method of any one of claims 73-95, wherein the change in the endogenous environment of the target macromolecule comprises a change in the level of molecular crowding.

97. The method of any one of claims 73-96, wherein the change in the endogenous environment comprises a reduction in the concentration of the key effectors of LLPS or phase transition.

98. The method of any one of claims 73-97, wherein the key effectors of LLPS or phase transition are scaffolding proteins or RNA.