Liquid-phase nucleic acid nanoparticles and their use

Abstract

A liquid-liquid phase separation system comprising nanostar droplets comprising nanostar condensates coated with a nucleic acid surfactant is provided. The nucleic acid surfactant forms a shell around the nanostar condensates, which prevents the nanostar condensates from adhering to hydrophobic surfaces. The size of the nanostar droplets is tunable simply by adjusting the nanostar: nucleic acid surfactant ratio to allow production of nanostar droplets having a radius in a range from about 30 nm to about 10 μm. Methods of using such nanostar condensates encapsulated with nucleic acid surfactants for delivery of a payload such as a drug, protein, nucleic acid, gene therapy agent, gene editing agent, or other molecule of interest are also provided.

Description

LIQUID-PHASE NUCLEIC ACID NANOPARTICLES AND THEIR USECROSS-REFERENCE TO ELATED APPLICATION

[0001] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63,530,316 filed August 2, 2023, the disclosure of which is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0002] Biomolecular condensates have a critical role in cellular function. As a tool to study properties of biomolecular condensates, synthetic biomolecules are widely used. The study of synthetic condensates will aid in understanding roles of membraneless organelles in cellular functioning. In particular, the ability to control biomolecular synthetic condensates would enhance the use of such condensates as common bioreactors and provide new drug delivery methods.SUMMARY OF THE INVENTION

[0003] A liquid-liquid phase separation system comprising nanostar droplets comprising nanostar condensates coated with a nucleic acid surfactant is provided. The nucleic acid surfactant forms a shell around the nanostar condensates, which prevents the nanostar condensates from adhering to hydrophobic surfaces. The size of nanostar droplets is tunable simply by adjusting the nanostar: nucleic acid surfactant ratio to allow production of nanostar droplets having a radius in a range from about 30 nm to about 10 pm. Methods of using such nanostar condensates encapsulated with nucleic acid surfactants for delivery of a payload such as a drug, protein, nucleic acid, gene therapy agent, gene editing agent, or other molecule of interest are also provided.

[0004] In one aspect, a composition is provided, the composition comprising: (a) a nanostar condensate comprising a plurality of DNA nanostars assembled from a plurality of single-stranded DNA oligonucleotides, wherein each DNA nanostar comprises: a core and a plurality of arms extending from the core, wherein each arm comprises i) a double-stranded region and ii) a singlestranded overhang region comprising a binding site for the single-stranded overhang region of an arm of another DNA nanostar, wherein the plurality of DNA nanostars assemble into a mesh structure in the nanostar condensate; and (b) a plurality of nucleic acid surfactant molecules, wherein each nucleic acid surfactant molecule comprises a double-stranded region and a single-stranded overhang region, wherein the single stranded overhang region of the nucleic acid surfactant moleculeis complementary to and hybridizes to the single-stranded overhang regions of the plurality of arms, wherein the plurality of nucleic acid surfactant molecules encapsulate the nanostar condensate.

[0005] In certain embodiments, the binding site comprises a palindromic sequence. In some embodiments, the palindromic sequence is at least 6 nucleotides in length. In some embodiments, the palindromic sequence is 6 nucleotides to 12 nucleotides in length.

[0006] In certain embodiments, each arm further comprises a linker comprising one or more unpaired bases between the double-stranded region and the binding site. In some embodiments, the linker is one to three nucleotides in length.

[0007] In certain embodiments, a DNA nanostar further comprises a linker comprising one or more unpaired bases between the core and the arm to increase the angular flexibility of an arm. In some embodiments, the linker is one to three nucleotides in length.

[0008] In certain embodiments, the nucleic acid surfactant comprises a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).

[0009] In certain embodiments, the nanostar condensate has a radius having a length in a range from about 30 nm to about 10 pm.

[0010] In certain embodiments, each DNA nanostar comprises 3 arms, 4 arms, 5 arms, or six arms.

[0011] In certain embodiments, the plurality of single-stranded DNA oligonucleotides used to assemble the DNA nanostars comprises 3 to 6 different single-stranded DNA oligonucleotides.

[0012] In certain embodiments, the nanostar condensate comprises a 3-arm nanostar comprising3 different single stranded DNA oligonucleotides.

[0013] In certain embodiments, the nanostar condensate comprises a 4-arm nanostar comprising4 different single stranded DNA oligonucleotides.

[0014] In certain embodiments, the nanostar condensate comprises a 5-arm nanostar comprising5 different single stranded DNA oligonucleotides.

[0015] In certain embodiments, the nanostar condensate comprises a 6-arm nanostar comprising6 different single stranded DNA oligonucleotides.

[0016] In certain embodiments, each DNA nanostar is assembled from a plurality of singlestranded DNA oligonucleotides, wherein the plurality of single-stranded DNA oligonucleotides comprises: a first single-stranded DNA oligonucleotide comprising a first core region, wherein the first core region is located between a first arm region and a second arm region; a second singlestranded DNA oligonucleotide comprising a second core region, wherein the second core region is located between a third arm region and a fourth arm region; a third single-stranded DNAoligonucleotide comprising a third core region, wherein the third core region is located between a fifth arm region and a sixth arm region; and a fourth single-stranded DNA oligonucleotide comprising a fourth core region, wherein the fourth core region is located between a seventh arm region and a eighth arm region; wherein the first arm region of the first single-stranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the third arm region of the second single-stranded DNA oligonucleotide to form a first double stranded region of a first nanostar arm, and ii) a sequence that is non-complementary to the third arm region that forms a first singlestranded overhang region of the first nanostar arm; wherein the second arm region of the first singlestranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the fifth arm region of the third single-stranded DNA oligonucleotide to form a second double stranded region of a second nanostar arm, and ii) a sequence that is non-complementary to the fifth arm region that forms a second single-stranded overhang region of the second nanostar arm; wherein the fourth arm region of the second single-stranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the seventh arm region of the fourth singlestranded DNA oligonucleotide to form a third double stranded region of a third nanostar arm, and ii) a sequence that is non-complementary to the seventh arm region that forms a third single-stranded overhang region of the third nanostar arm; and wherein the sixth arm region of the third singlestranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the eighth arm region of the fourth single-stranded DNA oligonucleotide to form a fourth double stranded region of a fourth nanostar arm, and ii) a sequence that is non-complementary to the eighth arm region that forms a fourth single-stranded overhang region of the fourth nanostar arm.

[0017] In certain embodiments, the nanostar condensate, encapsulated by the plurality of nucleic acid surfactant molecules, does not adhere to a hydrophobic surface.

[0018] In certain embodiments, the composition further comprises a payload, wherein the nanostar condensate encapsulated by the plurality of nucleic acid surfactant molecules contains the payload.

[0019] In certain embodiments, the payload is covalently linked to the plurality of nucleic acid surfactant molecules.

[0020] In certain embodiments, the payload is conjugated to a single stranded nucleic acid, wherein the single stranded nucleic acid comprises a region that is complementary to and hybridizes to a single stranded region of each nucleic acid surfactant molecule, thereby presenting the payload on the plurality of nucleic acid surfactant molecules encapsulating the nanostar condensate.

[0021] In certain embodiments, the payload is encapsulated by the nanostar condensate.

[0022] In certain embodiments, the payload comprises a therapeutic agent, a gene therapy agent, a gene editing agent, or a differentiation agent. In some embodiments, the payload comprises a protein (e.g., an antibody, a growth factor, a hormone, a neuropeptide, or an enzyme), a nucleic acid, a drug, an antisense oligonucleotide, an inhibitory RNA (e.g., a microRNA (miRNA), short interfering RNA (siRNA), or short hairpin RNA (shRNA)), a messenger RNA, a transgene, or an aptamer. In some embodiments, the payload is a gene editing agent such as, but not limited to, a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas endonuclease, a peptide-nucleic acid (PNA), a donor polynucleotide that can be used for homology-directed repair, a guide RNA, or a ribonucleoprotein comprising a Cas endonuclease complexed with a guide RNA (e.g., that targets the Cas endonuclease to a target gene of interest for gene editing).

[0023] In certain embodiments, the composition further comprises a plurality of targeting agents linked to the plurality of nucleic acid surfactant molecules. In some embodiments, the targeting agents are antibodies that specifically bind to a cell surface marker on a target cell.

[0024] In certain embodiments, the nucleic acid surfactant molecule is conjugated to a polymer. In some embodiments, the polymer is biodegradable. In some embodiments, the polymer is selected from the group consisting of polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA), poly(e-caprolactone) (POL), poly(lactide-co-glycolide) (PLGA), and a copolymer of poly(lactide-co-glycolide) and poly(e-caprolactone) (PLGA / PCL).

[0025] In certain embodiments, the composition further comprises a shell comprising a plurality of non-nucleic acid molecules. In some embodiments, the non-nucleic acid molecules are targeting agents or conjugated to targeting agents. In some embodiments, the non-nucleic acid molecules are selected from the group consisting of peptides, peptoids, peptide nucleic acids, aptamers, antibodies, and polymers. In some embodiments, the polymers are selected from the group consisting of polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA), poly(e- caprolactone) (PCL), poly(lactide-co-glycolide) (PLGA), and a copolymer of poly(lactide-co- glycolide) and poly(e-caprolactone) (PLGA / PCL). In some embodiments, the peptides are targeting peptides.

[0026] In another aspect, a method of delivering a payload into a cell is provided, the method comprising introducing a composition described herein into the cell.

[0027] In certain embodiments, the cell is an adult cell, a stem cell, or a progenitor cell.

[0028] In certain embodiments, the delivery is performed in vitro, ex vivo, or in vivo.

[0029] In certain embodiments, the payload comprises a therapeutic agent.

[0030] In another aspect, a method of decreasing the size of the nanostar condensate is provided, the method comprising increasing the concentration of the nucleic acid surfactant molecule or increasing the ratio of the nucleic acid surfactant molecule to the nanostar.

[0031] In another aspect, a method of delivering a therapeutic agent to a subject is provided, the method comprising administering to the subject a composition comprising a nanostar condensate encapsulated by the plurality of nucleic acid surfactant molecules carrying a payload comprising the therapeutic agent.

[0032] In another aspect, a method of genetically modifying a cell is provided, the method comprising introducing into the cell a composition comprising a nanostar condensate encapsulated by a plurality of nucleic acid surfactant molecules carrying a payload comprising a gene editing reagent.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0034] FIGS. 1A-1 B. (FIG. 1 A) Molecular profile of NS-surfactant system. (FIG. I B) Plot of fluorescence intensity radially from the center of a nanostar droplet to analyze the concentration of DNA surfactant across the droplet interface. 5% of the nanostars were tagged with a Cy5 fluorophore at a similar location on their first arm. We observed a ring of higher fluorescence intensity on the surface of the nanostar droplets under a confocal microscope, which indicates a higher DNA surfactant concentration on the surface of the nanostar droplets. We see a lower intensity within the droplet.

[0035] FIGS. 2A-2B. (FIG. 2A) Droplet images under confocal microscopy. The samples are 2 M nanostar with a concentration of DNA surfactant indicated in each image. (FIG. 2B) Droplet size distribution at low surfactant:NS ratio.

[0036] FIGS. 3A-3B. Droplet size distribution at high surfactant:NS ratio, as measured by dynamic light scattering (DLS) (FIG. 3A) and transmission electron microscopy (TEM) (FIG. 3B).

[0037] FIGS. 4A-4C. (FIG. 4A) Droplet images under confocal microscopy. The samples are 2 M nanostar with a concentration of DNA surfactant indicated in each image. (FIG. 4B) Droplet size distribution at low surfactant:NS ratio. (FIG. 4C) Diagrams of droplets on the surface.DETAILED DESCRIPTION OF THE INVENTION

[0038] A liquid-liquid phase separation system comprising nanostar droplets comprising nanostar condensates coated with a nucleic acid surfactant is provided. The nucleic acid surfactant forms a shell around the nanostar condensates, which prevents the nanostar condensates from adhering to hydrophobic surfaces. The size of the nanostar droplets is tunable simply by adjusting the nanostar: nucleic acid surfactant ratio to allow production of nanostar droplets having a radius in a range from about 30 nm to about 10 pm. Methods of using such nanostar condensates encapsulated with nucleic acid surfactants for delivery of a payload such as a drug, protein, nucleic acid, gene therapy agent, gene editing agent, or other molecule of interest are also provided.

[0039] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0040] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0042] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0043] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a droplet" includes a plurality of such droplets and reference to "the nucleic acid" includes reference to one or more nucleic acids and equivalents thereof, e.g., polynucleotides, oligonucleotides, and the like, known to those skilled in the art, and so forth.

[0044] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.Definitions

[0045] The term "about," particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.

[0046] By “subject” is meant any member of the subphylum Chordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.

[0047] The term “DNA nanostar” refers to a multiarmed DNA structure produced from selfassembly of a plurality of single-stranded DNA oligonucleotides. The sequences of the DNA oligonucleotides are designed to self-assemble to form structures having a core region and multiple double-stranded arms with “sticky” single stranded overhang regions at the end of each arm. The “sticky” single stranded overhang region of each arm comprises a recognition sequence to allow hybridization to the sticky end of another nanostar, wherein nanostars assemble into a mesh-like structure. In some cases, the recognition sequence in the single stranded overhang region at the end of an arm comprises a palindromic sequence. An arm may further comprise a linker sequencecomprising one or more unpaired nucleotides between the double stranded portion of the arm and the single stranded recognition sequence, which increases flexibility at the junction between the arm and the region that binds to another nanostar. In addition, a nanostar may further comprising a linker comprising one or more unpaired nucleotides between the core region and the arm region to increase the angular flexibility of an arm. Such linker sequences typically range from one nucleotide to a few nucleotides in length. The number of arms may vary depending on the sequences of the DNA oligonucleotides and the number of different DNA oligonucleotides used to assemble the nanostar. In some embodiments, a DNA nanostar has 3 arms, 4 arms, 5 arms, or 6 arms. For example, three single-stranded DNA oligonucleotides hybridize to form a nanostar with three arms; four singlestranded DNA oligonucleotides hybridize to form a nanostar with four arms; five single-stranded DNA oligonucleotides hybridize to form a nanostar with five arms; and six single-stranded DNA oligonucleotides hybridize to form a nanostar with six arms, and so on. For a further description of DNA nanostars, see, e.g., Biffi et al. (2013) Proc Natl Acad Sci USA 110(39):15633-7, Conrad et al. (2019) Proc Natl Acad Sci USA 116(15):7238-7243, Lattuada et al. (2020) Nanoscale 12(45):23003- 23012, Lee et al. (2021 ) Nanoscale 13(41 ):17638-17647, Nguyen and Saleh (2017) Soft Matter 13(32):5421 -5427, and Jeon et al. (2018) Soft Matter 14(34):7009-7015; herein incorporated by reference in their entireties.

[0048] “Diameter” as used in reference to a shaped structure (e.g., nanostar condensate, droplet, particle, cell, etc.) refers to a length that is representative of the overall size of the structure. The length may in general be approximated by the diameter of a circle or sphere that circumscribes the structure.

[0049] By "isolated" is meant, when referring to a polypeptide or peptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro molecules of the same type. The term "isolated" with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.

[0050] The terms “treat”, “treating”, treatment,” “prevent,” “preventing,” and the like refer to a course of action (such as administering an agent or a pharmaceutical composition comprising an agent) initiated after a disease, disorder or condition, or a symptom thereof, has been diagnosed, observed, and the like so as to eliminate, reduce, suppress, mitigate, or ameliorate, either temporarily or permanently, at least one of the underlying causes of a disease, disorder, or condition afflicting asubject, or at least one of the symptoms associated with a disease, disorder, or condition afflicting a subject. Thus, treatment includes inhibiting (i.e., arresting the development or further development of the disease, disorder or condition or clinical symptoms association therewith) an active disease.

[0051] The term “in need of treatment” as used herein refers to a judgment made by a physician or other caregiver that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of the physician’s or caregiver's expertise.

[0052] The phrase “therapeutically effective amount” refers to the administration of an agent to a subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to a patient. The therapeutically effective amount can be ascertained by measuring relevant physiological effects.

[0053] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0054] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0055] The term "antibody" encompasses monoclonal antibodies and polyclonal antibodies, as well as hybrid antibodies, altered antibodies, chimeric antibodies, and humanized antibodies. The term antibody includes: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991 ) Nature 349:293-299; and U.S. Pat. No. 4,816,567); P(ab')2 and F(ab) fragments; Fvmolecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659- 2662; and Ehrlich et al. (1980) Biochem 19:4091 -4096); single-chain Fv molecules (scFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanobodies or single-domain antibodies (sdAb) (see, e.g., Wang et al. (2016) Int J Nanomedicine 1 1 :3287-3303, Vincke et al. (2012) Methods Mol Biol 911 : 15-26; dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Packet al. (1992) Biochem 31 :1579-1584; Cumber et al. (1992) J Immunology 14913:120-126); humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain specificbinding properties of the parent antibody molecule.

[0056] The terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" are used herein to include a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded DNA, as well as triple-, double- and single-stranded RNA. It also includes modifications, such as by methylation and / or by capping, and unmodified forms of the polynucleotide. More particularly, the terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide which is an N- or C- glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. There is no intended distinction in length between the terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule," and these terms will be used interchangeably. Thus, these terms include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3' P5' phosphoramidates, 2'-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and singlestranded RNA, microRNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels which are known in the art, methylation, "caps," substitution of one or more of the naturally occurring nucleotides with an analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5- propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylguanine, and 2-thiocytidine), internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies,signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide. The term also includes locked nucleic acids (e.g., comprising a ribonucleotide that has a methylene bridge between the 2'-oxygen atom and the 4'-carbon atom). See, for example, Kurreck et al. (2002) Nucleic Acids Res. 30: 191 1 -1918; Elayadi et al. (2001 ) Curr. Opinion Invest. Drugs 2: 558-561 ; Orum et al. (2001) Curr. Opinion Mol. Ther. 3: 239-243; Koshkin et al. (1998) Tetrahedron 54: 3607-3630; Obika et al. (1998) Tetrahedron Lett. 39: 5401 -5404.

[0057] The terms "hybridize" and "hybridization" refer to the formation of complexes between nucleotide sequences which are sufficiently complementary to form complexes via Watson-Crick base pairing.

[0058] The term “hybridization conditions” as used herein refers to conditions that allow hybridization of a nucleic acid to a complementary nucleic acid, e.g., a nucleic acid surfactant attached to a nanostar condensate may specifically bind to a complementary nucleic acid of a nanostar via Watson-Crick base pairing under hybridization conditions.

[0059] The term "homologous region" refers to a region of a nucleic acid with homology to another nucleic acid region. Thus, whether a "homologous region" is present in a nucleic acid molecule is determined with reference to another nucleic acid region in the same or a different molecule. Further, since a nucleic acid is often double-stranded, the term "homologous, region," as used herein, refers to the ability of nucleic acid molecules to hybridize to each other. For example, a single-stranded nucleic acid molecule can have two homologous regions which are capable of hybridizing to each other. Thus, the term "homologous region" includes nucleic acid segments with complementary sequences. Homologous regions may vary in length, but will typically be between 4 and 500 nucleotides (e.g., from about 4 to about 40, from about 40 to about 80, from about 80 to about 120, from about 120 to about 160, from about 160 to about 200, from about 200 to about 240, from about 240 to about 280, from about 280 to about 320, from about 320 to about 360, from about 360 to about 400, from about 400 to about 440, etc.).

[0060] As used herein, the terms "complementary" or "complementarity" refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in theart are aware, when using RNA as opposed to DNA, uracil (II) rather than thymine (T) is the base that is considered to be complementary to adenosine. However, when a uracil is denoted in the context of the present invention, the ability to substitute a thymine is implied, unless otherwise stated. "Complementarity" may exist between two RNA strands, two DNA strands, or between a RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be "complementary" and able to form a duplex despite having less than perfect or less than 100% complementarity. Two sequences are "perfectly complementary" or "100% complementary" if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region. Two or more sequences are considered "perfectly complementary" or "100% complementary" even if either or both polynucleotides contain additional non-complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other. "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter of ordinary skill in the art.

[0061] For purposes of Cas9 targeting, a guide RNA (gRNA) may comprise a sequence "complementary" to a target sequence (e.g., major or minor allele), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.

[0062] A "target site" or "target sequence" is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a therapeutic agent, a guide RNA (gRNA), or a homology arm of a donor polynucleotide. The target site may be allele-specific (e.g., a major or minor allele).

[0063] The term "donor polynucleotide" refers to a polynucleotide that provides a sequence of an intended edit to be integrated into the genome at a target locus by homology-directed repair (HDR).

[0064] By "homology arm" is meant a portion of a donor polynucleotide that is responsible for targeting the donor polynucleotide to the genomic sequence to be edited in a cell. The donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence comprising the intended edit to the genomic DNA. The homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relativeposition of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively. The nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.

[0065] The term "Cas9" as used herein encompasses type II clustered regularly interspaced short palindromic repeats (CRISPR) system Cas9 endonucleases from any species, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks). A Cas9 endonuclease binds to and cleaves DNA at a site comprising a sequence complementary to its bound guide RNA (gRNA).

[0066] A Cas9 polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP 002989955, WP 038434062, WP 011528583); Campylobacter jejuni (WP 022552435, YP_002344900), Campylobacter coli (WP_060786116); Campylobacter fetus (WP 059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP 033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861 ); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus torquisl (NC_018721 ); Streptococcus thermophilus (YP 820832), Streptococcus mutans (WP_061046374, WP_024786433); Listeria innocua (NP_472073); Listeria monocytogenes (WP 061665472); Legionella pneumophila (WP 062726656); Staphylococcus aureus (WP 001573634); Francisella tularensis (WP 032729892, WP_014548420), Enterococcus faecalis (WP 033919308); Lactobacillus rhamnosus (WP 048482595, WP 032965177); and Neisseria meningitidis (WP 061704949, YP 002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can beused for genome editing, as described herein, wherein the variant retains biological activity, such as Cas9 site-directed endonuclease activity. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol. 198(5):797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385-397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10):6091 -6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[0067] The terms “polypeptide,” “peptide,” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified polypeptide backbones. The terms include fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusion proteins with heterologous and homologous leader sequences, with or without N-terminus methionine residues; immunologically tagged proteins; and the like.

[0068] As used herein, the term "binding pair" refers to first and second molecules that specifically bind to each other. The binding between the members of the binding pair is typically noncovalent.

[0069] The terms “specific binding,” “specifically binds,” and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction. In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a KD(dissociation constant) of 10'5M or less (e.g., 10‘6M or less, W7M or less, 10'8M or less, 10-9M or less, 10‘10M or less, 10‘11M or less, 10‘12M or less). "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower KD. In an embodiment, affinity is determined by surface plasmon resonance (SPR), e.g., as used by Biacore systems. The affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, e.g., at 25°C.Liquid-Liquid Phase Separation System Comprising a Nanostar Condensate

[0070] A liquid-liquid phase separation system comprising nanostar droplets is provided. In some embodiments, the nanostar droplets comprise a DNA nanostar condensate encapsulated with a nucleic acid surfactant to form a micelle-like structure. The term “DNA nanostar” refers to a multiarmed DNA structure produced from self-assembly of a plurality of single-stranded DNA oligonucleotides. The sequences of the DNA oligonucleotides are designed to self-assemble to form structures having a core region and multiple double-stranded arms with “sticky” single stranded overhang regions at the end of each arm. The “sticky” single stranded overhang region at the end of each arm comprises a recognition sequence to allow hybridization to the sticky end of the arm ofanother nanostar such that the nanostars assemble into a mesh-like structure. In some embodiments, the recognition sequence comprises a palindromic sequence. An arm may further comprise a linker sequence comprising one or more unpaired nucleotides between the double stranded portion of the arm and the single stranded recognition sequence, which increases flexibility at the junction between the arm and the region that binds to another nanostar. In addition, a nanostar may further comprising a linker comprising one or more unpaired nucleotides between the core region and the arm region to increase the angular flexibility of an arm. Such linker sequences typically range from one nucleotide to a few nucleotides in length. The number of arms may vary depending on the sequences of the DNA oligonucleotides in the composition and the number of different DNA oligonucleotides used to assemble the nanostar. In some embodiments, a DNA nanostar has 3 arms, 4 arms, 5 arms, or 6 arms, or more. For example, three single-stranded DNA oligonucleotides hybridize to form a nanostar with three arms; four single-stranded DNA oligonucleotides hybridize to form a nanostar with four arms; five single-stranded DNA oligonucleotides hybridize to form a nanostar with five arms; and six single-stranded DNA oligonucleotides hybridize to form a nanostar with six arms, and so on. For a further description of DNA nanostars, see, e.g., Biffi et al. (2013) Proc Natl Acad Sci USA 110(39):15633-7, Conrad et al. (2019) Proc Natl Acad Sci USA 116(15):7238- 7243, Lattuada et al. (2020) Nanoscale 12(45):23003-23012, Lee et al. (2021 ) Nanoscale 13(41 ):17638-17647, Nguyen and Saleh (2017) Soft Matter 13(32):5421 -5427, and Jeon et al. (2018) Soft Matter 14(34):7009-7015; herein incorporated by reference in their entireties.

[0071] The diameter of a nanostar droplet comprising a nanostar condensate encapsulated by a nucleic acid surfactant refers to the length from one end of the nanostar droplet to the diametrically opposite end and may range from nanometers to micrometers. The size of the nanostar droplets may be controlled by the amount of the nucleic acid surfactant and / or the nanostannucleic acid surfactant ratio used to form the nanostar droplets. In some embodiments, a nanostar droplet has a radius having a length in a range from about 30 nm to about 10 pm, including any length in between such as 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, or 10 pm, as measured by dynamic light scattering or transmission electron microscopy (TEM).

[0072] The nucleic acid surfactant forms a shell around the nanostar condensates, which prevents the nanostar condensates from adhering to hydrophobic surfaces. In certain embodiments, the nanostar: nucleic acid surfactant ratio is in a range from 1 :5 to 5:1 , 1 :4 to 4:1 , 1 :3 to 3:1 , 1 :2 to2:1 , or 1 :3 to 1 :1 , or any ratio within these ranges such as 1 :5, 1 :4, 1 :3, 1 :2, 1 :1 , 2:5, 2:3, 2:1 , 3:5, 3:4, 3:2, 3:1 , 4:5, 4:3, 4:1 , 5:4, 5:3, 5:2, or 5:1 .

[0073] In certain embodiments, a single-stranded DNA oligonucleotide, used to assemble a nanostar, has a length in a range from 20 nucleotides to 80 nucleotides, 30 nucleotides to 60 nucleotides, 40 nucleotides to 50 nucleotides including any length within these ranges such as 20, 21 , 22, 23 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, or 80 nucleotides in length.

[0074] Each single-stranded DNA oligonucleotide, used in assembly of a nanostar, is designed with a non-binding core center region that separates the two arm regions, wherein each arm region hybridizes with an arm region of a different single-stranded DNA oligonucleotide. The arm regions of two single-stranded DNA oligonucleotides, which hybridize to each other, may include a contiguous stretch of at least 4 bases that are complementary to each other. In some embodiments, the arm regions of two single-stranded DNA oligonucleotides, which hybridize to each other, include a plurality of contiguous stretches of at least 4 bases that are complementary to each other, which contiguous stretches are separated by bases that are not complementary to each other. In certain embodiments, the region of complementarity is about 10-50 bases, 20-40 bases, 20-35 bases, 20- 30 bases, or 20-26 bases. In certain embodiments, the contiguous stretch of bases is less than 100% complementary. For example, in a stretch of at least 20 bases, there may be at least 99% complementarity, 98% complementarity, 97% complementarity, 96% complementarity, or 95% complementarity. In certain embodiments, the region of complementarity may be about 10-50 bases, 20-40 bases, 20-35 bases, 20-30 bases, or 20-26 bases, wherein all of the bases are complementary.

[0075] In certain embodiments, the double-stranded arm region of a nanostar has a length ranging from 10 base pairs (bp) to 50 bp, 15 bp to 40 bp, or 20 bp to 30 bp, including any length within these ranges such as 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 28 bp, 29 bp, 30 bp, 31 bp, 32 bp, 33 bp, 34 bp, 35 bp, 36 bp, 37 bp, 38 bp, 39 bp, 40 bp, 41 bp, 42 bp, 43 bp, 44 bp, 45 bp, 46 bp, 47 bp, 48 bp, 49 bp, or 50 bp in length.

[0076] In certain embodiments, the single stranded overhang region at the end of each arm has a length ranging from 6 nucleotides to 20 nucleotides, 7 nucleotides to 15 nucleotides, or 7 nucleotides to 10 nucleotides in length, including any length within these ranges such as 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.

[0077] In certain embodiments, each DNA nanostar is assembled from a plurality of singlestranded DNA oligonucleotides, wherein the plurality of single-stranded DNA oligonucleotidescomprises: a first single-stranded DNA oligonucleotide comprising a first core region, wherein the first core region is located between a first arm region and a second arm region; a second singlestranded DNA oligonucleotide comprising a second core region, wherein the second core region is located between a third arm region and a fourth arm region; a third single-stranded DNA oligonucleotide comprising a third core region, wherein the third core region is located between a fifth arm region and a sixth arm region; and a fourth single-stranded DNA oligonucleotide comprising a fourth core region, wherein the fourth core region is located between a seventh arm region and a eighth arm region; wherein the first arm region of the first single-stranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the third arm region of the second single-stranded DNA oligonucleotide to form a first double stranded region of a first nanostar arm, and ii) a sequence that is non-complementary to the third arm region that forms a first singlestranded overhang region of the first nanostar arm; wherein the second arm region of the first singlestranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the fifth arm region of the third single-stranded DNA oligonucleotide to form a second double stranded region of a second nanostar arm, and ii) a sequence that is non-complementary to the fifth arm region that forms a second single-stranded overhang region of the second nanostar arm; wherein the fourth arm region of the second single-stranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the seventh arm region of the fourth singlestranded DNA oligonucleotide to form a third double stranded region of a third nanostar arm, and ii) a sequence that is non-complementary to the seventh arm region that forms a third single-stranded overhang region of the third nanostar arm; and wherein the sixth arm region of the third singlestranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the eighth arm region of the fourth single-stranded DNA oligonucleotide to form a fourth double stranded region of a fourth nanostar arm, and ii) a sequence that is non-complementary to the eighth arm region that forms a fourth single-stranded overhang region of the fourth nanostar arm.

[0078] The nucleic acid surfactant molecule comprises a polymer of naturally occurring or modified ribonucleotides or deoxyribonucleotides having a length of at least 100 bp. In some embodiments, the nucleic acid surfactant molecule contains one or more chemical modifications, such as, but not limited to, locked nucleic acids, peptide nucleic acids, sugar modifications, such as 2'-O-alkyl (e.g., 2'-O-methyl, 2'-0-methoxyethyl), 2'-fluoro, and 4'-thio modifications, and backbone modifications, such as one or more phosphorothioate, morpholino, or phosphonocarboxylate linkages.

[0079] Each nucleic acid surfactant molecule comprises a double-stranded region and a singlestranded overhang region with a sticky end, wherein the single stranded overhang region of the nucleic acid surfactant molecule is complementary to and hybridizes to at least a portion of the singlestranded overhang regions of the nanostar arms. In some embodiments, the single stranded overhang region of a nucleic acid surfactant is designed to be complimentary to an entire single stranded overhang sequence of a nanostar arm. For example, the single stranded overhang region of the nucleic acid surfactant molecule may have 100% complementarity, 99% complementarity, 98% complementarity, 97% complementarity, 96% complementarity, or 95% complementarity to the single stranded overhang region of a nanostar arm.

[0080] The nucleic acid surfactant molecules are sufficiently long to impede penetration into the interior of the nanostar droplet mesh such that when nucleic acid surfactant molecules are mixed with a nanostar droplet, the nucleic acid surfactant molecules bind to the surface of the nanostar droplet mesh to form a shell surrounding the nanostar condensate. In some embodiments, the double-stranded region of the nucleic acid surfactant has a length ranging from 100 bp to 1000 bp, 200 bp to 800 bp, 300 bp to 600 bp, or 400 bp to 500 bp, including any length within these ranges such as 100 bp, 150 bp, 200 bp, 220 bp, 240 bp, 260 bp, 280 bp, 300 bp, 320 bp, 340 bp, 360 bp, 380 bp, 400 bp, 420 bp, 440 bp, 460 bp, 480 bp, 500 bp, 520 bp, 540 bp, 560 bp, 580 bp, or 600 bp in length. In one embodiment, the nucleic acid surfactant has a length of 405 bp.

[0081] In some embodiments, the nucleic acid surfactant molecule is conjugated to a polymer, such as a biocompatible, non-immunogenic, and / or biodegradable polymer. In certain cases, the polymer is polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA), and / or poly(e- caprolactone) (POL). In certain embodiments, the polymer is a copolymer such as poly(lactide-co- glycolide) (PLGA) or poly(lactide-co-glycolide) poly(e-caprolactone) (PLGA / PCL).

[0082] In some embodiments, a nanostar condensate is encapsulated with a shell comprising molecules other than nucleic acids. In some embodiments, the non-nucleic acid molecules included in the shell are of sufficient size to impede penetration into the interior of the nanostar droplet mesh. In some embodiments, the non-nucleic acid molecules included in the shell are sufficiently hydrophilic to prevent adherence of the nanostar droplet to a hydrophobic surface. Exemplary non- nucleic acid molecules that may be included in the shell include, but are not limited to, peptides, peptoids, peptide nucleic acids, aptamers, and polymers such as PEG, PLA, PGA, and / or PCL, and / or copolymers such as PLGA and / or PLGA / PCL. In some embodiments, a non-nucleic acid molecule of the shell is used as a targeting agent or is conjugated to a targeting agent to allow the nanostar particle to be used for targeted delivery of a payload to a target cell or tissue. For example,the shell may comprise a peptide, peptoid, peptide nucleic acid, antibody, or aptamer, which is a ligand that binds selectively to a cellular marker or a receptor on a target cell. In another example, a non-nucleic acid molecule of the shell is covalently linked to a targeting agent such as an antibody specific for an epitope on a cell marker or receptor of a target cell or tissue to provide targeted delivery. In some embodiments, targeting peptides, peptoids, peptide nucleic acids, or aptamers are selected based on the screening of combinatorial libraries. A single targeting agent may be used alone to provide selective targeting of a target cell or tissue or in combination with other targeting agents with different specificities to provide a multifunctional delivery system.Delivery of a Payload

[0083] Compositions comprising nanostar droplets comprising nanostar condensates encapsulated by nucleic acid surfactant molecules and / or other molecules can be used as carriers for delivery of a payload such as a drug, protein, nucleic acid, aptamer, gene therapy agent (e.g., recombinant polynucleotide encoding a therapeutic agent), gene editing agent, or other molecule of interest. The payload may include, without limitation, a protein such as an antibody, a growth factor, a hormone, a neuropeptide, or an enzyme; an inhibitory RNA such as a microRNA (miRNA), short interfering RNA (siRNA), or short hairpin RNA (shRNA); a mRNA, a transgene, an antisense oligonucleotide, an aptamer, a tRNA suppressor, a differentiation agent, or a gene editing agent such as a site-specific endonuclease (e.g., a clustered regularly interspaced short palindromic repeats (CRISPR)ZCRISPR-associated (Cas) nuclease, meganuclease, zinc-finger nuclease (ZFN), or transcription activator-like effector nuclease (TALEN), a donor polynucleotide that can be used for homology-directed repair for gene editing, a guide RNA, or a ribonucleoprotein comprising a Cas endonuclease complexed with a guide RNA. In some cases, a nanostar condensate, encapsulated by a plurality of nucleic acid surfactant molecules and / or other molecules, is used as a carrier for delivery of multiple payloads. Any combination of payloads disclosed herein is contemplated.

[0084] In some embodiments, the payload is encapsulated within the nanostar condensate. Other embodiments make use of the nucleic acid surfactant encapsulating the nanostar condensate for attaching molecules for delivery. For example, the nucleic acid surfactant can be used to deliver various therapeutic agents including nucleic acids that have complementary sequences to a portion of the nucleic acid surfactant. Examples include, without limitation, nucleic acids for use in gene therapy, antisense oligonucleotides, and inhibitory RNAs (e.g., miRNAs, siRNAs, and shRNAs) that have complementary sequences to the nucleic acid surfactant. A Cas9-guide RNA complex can hybridize to a sequence of the nucleic acid surfactant having a sequence that is complementary tothe guide RNA. In some embodiments, the nucleic acid surfactant comprises a homopolymer sequence, such as poly A, poly T, poly G, poly C, poly U, poly dA, poly dT, poly dG, poly dC, or poly dll sequence, which can be used to bind therapeutic agents having a complementary homopolymer sequence. For example, mRNA having a poly A tail can be attached to a nucleic acid surfactant having a poly T or poly U sequence. Other therapeutic agents such as proteins and antibodies may be coupled to nucleic acids complementary to the nucleic acid surfactant to allow hybridization for attachment to the carrier.

[0085] Alternatively, the payload may be attached to the nucleic acid surfactant or a nanostar of the carrier covalently. In certain embodiments, the payload is covalently attached to the nucleic acid surfactant or nanostar via a reactive group. Any suitable bioconjugation method may be used for conjugating a payload to a nucleic acid surfactant or nanostar. Crosslinking agents that can be used include, but are not limited to, dimethyl suberimidate, N-hydroxysuccinimide, formaldehyde, and glutaraldehyde. In addition, carboxyl-reactive chemical groups such as diazomethane, diazoacetyl, and carbodiimide can be included for crosslinking carboxylic acids to primary amines. In particular, the carbodiimide compounds, 1 -ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N',N'-dicyclohexyl carbodiimide (DCC) can be used for conjugation with carboxylic acids. In order to improve the efficiency of crosslinking reactions, N-hydroxysuccinimide (NHS) or a water- soluble analog (e.g., Sulfo-NHS) may be used in combination with a carbodiimide compound. The carbodiimide compound (e.g., EDC or DCC) couples NHS to carboxyl groups to form an NHS ester intermediate, which readily reacts with primary amines at physiological pH. For a description of various crosslinking agents and techniques, see, e.g., Wong and Jameson Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation (CRC Press, 2ndedition, 2011 ), Hermanson Bioconjugate Techniques (Academic Press, 3rdedition, 2013), herein incorporated by reference in their entireties.

[0086] The payload may be conjugated to a nucleic acid surfactant or nanostar via a covalent bond directly or indirectly. For example, the nucleic acid surfactant may be modified on its 3’-end or 5’-end by attachment of a reactive group which may react with a suitably modified payload to form a covalent bond between the nucleic acid surfactant and the payload. One or both strands of the nucleic acid surfactant may be modified to carry a payload. In some embodiments, the sense strand of the nucleic acid surfactant has a 5’-overhang region on one end that binds to a nanostar arm and is modified with a reactive group at the 3’-end, which reacts with a reactive group on the payload to covalently attach the nucleic acid surfactant to the payload. For example, the nucleic acid surfactant or nanostar may be modified to include an amino group, and the payload may be modified to includea carboxyl group which may react to covalently attach the payload to the nucleic acid surfactant or nanostar. Alternatively, the nucleic acid surfactant or nanostar may be modified to include a thiol group, and the payload may be modified to include a maleimide group which may react to covalently attach the payload to the nucleic acid surfactant or nanostar. In some embodiments, covalent attachment between the payload and the nucleic acid surfactant or nanostar may be achieved indirectly via a linker. For example, the nucleic acid surfactant or nanostar may be modified to include an amino group, which reacts with a carboxyl group in the linker. Alternatively, the linker may include a maleimide group to react with a thiol group in the nucleic acid surfactant or nanostar.

[0087] In certain embodiments, a payload is crosslinked to the nucleic acid surfactant or a nanostar of the carrier using click chemistry. Crosslinking with click chemistry can be performed with suitable crosslinking agents comprising reactive azide or alkyne functional groups. See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31 ; Evans, 2007, Aust J Chem 60:384-95; Millward et al. (2013) Integr Biol (Camb) 5(1 ):87-95, Lallana et al. (2012) Pharm Res 29(1 ):1 -34, Gregoritza et al. (2015) Eur J Pharm Biopharm. 97(Pt B):438-453, Musumeci et al. (2015) Curr Med Chem. 22(17):2022-2050, McKay et al. (2014) Chem Biol 21 (9):1075-1101 , Ulrich et al. (2014) Chemistry 20(1 ):34-41 , Pasini (2013) Molecules 18(8):9512-9530, and Wangler et al. (2010) Curr Med Chem. 17(1 1):1092-11 16; herein incorporated by reference in their entireties.

[0088] In particular, crosslinking can be performed using strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry, a Cu-free variation of click chemistry that is generally biocompatible with cells. SPAAC utilizes a substituted cyclooctyne having an internal alkyne in a strained ring system. Ring strain together with electron-withdrawing substituents in the cyclooctyne promote a [3+2] dipolar cycloaddition with an azide functional group. SPAAC can be used for bioconjugation and crosslinking by attaching azide and cyclooctyne moieties to molecules. For a description of SPAAC, see, e.g., Baskin et al. (2007) Proc Natl Acad Sci USA 104(43):16793-16797, Agard et al. (2006) ACS Chem. Biol. 1 : 644-648, Codelli et al. (2008) J. Am. Chem. Soc. 130:11486- 11493, Gordon et al. (2012) J. Am. Chem. Soc. 134:9199-9208, Jiang et al. (2015) Soft Matter 11 (30):6029-6036, Jang et al. (2012) Bioconjug Chem. 23(11 ):2256-2261 , Ornelas et al. (2010) J Am Chem Soc. 132(11 ):3923-3931 ; herein incorporated by reference in their entireties.

[0089] In certain embodiments, the payload is targeted to a specific cell or tissue type by conjugating the nanostar carrier to a targeting agent (e.g., for targeted delivery of a therapeutic agent to a specific site in the body for treatment of a disease). Suitable targeting agents include peptides, proteins, antibodies, antigen receptors, peptoids, peptide nucleic acids, aptamers, known cellsurface marker ligands, or any other ligand that binds specifically to a cell surface marker or areceptor on a target cell or tissue. Targeting agents can be attached to nanostar carriers in various ways. For example, a targeting agent can be attached covalently to the nucleic acid surfactant or a nanostar nucleotide of the carrier. Alternatively, a targeting agent can be attached to a non-nucleic acid molecule or incorporated into the outer shell of the carrier, as described above. Any suitable bioconjugation method can be used, including, without limitation, conjugation of maleimide with thiols, condensation of carboxyls with amines mediated by NHS / EDC, and various click-chemistry reactions, as described above. In other embodiments, targeting agents are attached to the nanostar carrier non-covalently through electrostatic interactions. For example, a targeting agent having groups with opposite charges to surface groups on the nanostar carrier can bind non-covalently to the nanostar carrier through ionic interactions.

[0090] In other embodiments, a binding pair is used to mediate attachments of targeting agents to the nanostar carrier. Exemplary binding pairs include any haptenic or antigenic compound in combination with a corresponding antibody or binding portion or fragment thereof (e.g., digoxigenin and an anti-digoxigenin antibody, fluorescein and an anti-fluorescein antibody, fluorescein isothiocyanate (FITC) and an anti-FITC antibody, dinitrophenol and an anti-dinitrophenol antibody, bromodeoxyuridine and an anti-bromodeoxyuridine antibody), an epitope tag and corresponding antibody or binding portion or fragment thereof (e.g., FLAG tag / anti-FLAG antibody), antibodies bound to other antibodies (e.g., mouse immunoglobulin and goat anti-mouse immunoglobulin), and antibodies bound to antibody-binders like protein A / G; and nonimmunological binding pairs (e.g., biotin-avidin, biotin-streptavidin, His-tag / histidine multimer, hormone (e.g., thyroxine and cortisol)- hormone binding protein, receptor-receptor agonist or antagonist (e.g., acetylcholine receptor- acetylcholine or an analog thereof), lectin-carbohydrate, enzyme-enzyme cofactor, enzyme-enzyme- inhibitor, and complementary polynucleotide pairs capable of forming nucleic acid duplexes) and the like. It is to be understood that the invention is not limited in this regard and one of ordinary skill is able to substitute these affinity pairs with other binding pairs.

[0091] The targeting agent is functionalized with one member of a binding pair while a surface of the nanostar carrier is functionalized with the other member of the binding pair. For example, biotinylated, imino-biotinylated, or desthio-biotinylated targeting agents can bind to a nanostar carrier functionalized with streptavidin, avidin, or a derivatives thereof (e.g., neutravidin); targeting agents functionalized with digoxigenin can bind to a nanostar carrier functionalized with an anti-digoxigenin antibody; targeting agents functionalized with a FLAG tag can bind to a nanostar carrier functionalized with an anti-FLAG antibody; and targeting agents functionalized with a His-tag can bind to a nanostar carrier functionalized with a histidine multimer; and so on.

[0092] Other embodiments described herein will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All U.S. Patents and other references noted herein for whatever reason are specifically incorporated by reference. The specification and examples should be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.Examples of Non-Limiting Aspects of the Disclosure

[0093] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-52 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:1 . A composition comprising:(a) a nanostar condensate comprising a plurality of DNA nanostars assembled from a plurality of single-stranded DNA oligonucleotides, wherein each DNA nanostar comprises: a core and a plurality of arms extending from the core, wherein each arm comprises i) a double-stranded region and ii) a single-stranded overhang region comprising a binding site for the single-stranded overhang region of an arm of another DNA nanostar, wherein the plurality of DNA nanostars assemble into a mesh structure in the nanostar condensate; and(b) a plurality of nucleic acid surfactant molecules, wherein each nucleic acid surfactant molecule comprises a double-stranded region and a single-stranded overhang region, wherein the single stranded overhang region of the nucleic acid surfactant molecule is complementary to and hybridizes to the single-stranded overhang regions of the plurality of arms, wherein the plurality of nucleic acid surfactant molecules encapsulate the nanostar condensate.2. The composition of aspect 1 , wherein the binding site comprises a palindromic sequence.3. The composition of aspect 2, wherein the palindromic sequence is 6 nucleotides to 12 nucleotides in length.4. The composition of any one of aspects 1 -3, wherein each arm further comprises a linker comprising one or more unpaired bases between the double-stranded region and the binding site.5. The composition of aspect 4, wherein the linker is one to three nucleotides in length.6. The composition of any one of aspects 1 -5, wherein the nucleic acid surfactant comprises a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).7. The composition of any one of aspects 1 -6, wherein the nanostar condensate has a radius having a length in a range from about 30 nm to about 10 pm.8. The composition of any one of aspects 1 -7, wherein each DNA nanostar comprises 3 arms, 4 arms, 5 arms, or six arms.9. The composition of any one of aspects 1 -8, wherein the plurality of single-stranded DNA oligonucleotides comprises 3 to 6 different single-stranded DNA oligonucleotides.10. The composition of aspect 9, wherein the nanostar condensate comprises a 3-arm nanostar comprising 3 different single stranded DNA oligonucleotides.11 . The composition of aspect 9, wherein the nanostar condensate comprises a 4-arm nanostar comprising 4 different single stranded DNA oligonucleotides.12. The composition of aspect 1 1 , wherein each DNA nanostar is assembled from a plurality of single-stranded DNA oligonucleotides, wherein the plurality of single-stranded DNA oligonucleotides comprises: a first single-stranded DNA oligonucleotide comprising a first core region, wherein the first core region is located between a first arm region and a second arm region; a second single-stranded DNA oligonucleotide comprising a second core region, wherein the second core region is located between a third arm region and a fourth arm region;a third single-stranded DNA oligonucleotide comprising a third core region, wherein the third core region is located between a fifth arm region and a sixth arm region; and a fourth single-stranded DNA oligonucleotide comprising a fourth core region, wherein the fourth core region is located between a seventh arm region and a eighth arm region; wherein the first arm region of the first single-stranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the third arm region of the second singlestranded DNA oligonucleotide to form a first double stranded region of a first nanostar arm, and ii) a sequence that is non-complementary to the third arm region that forms a first single-stranded overhang region of the first nanostar arm; wherein the second arm region of the first single-stranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the fifth arm region of the third single-stranded DNA oligonucleotide to form a second double stranded region of a second nanostar arm, and ii) a sequence that is non-complementary to the fifth arm region that forms a second single-stranded overhang region of the second nanostar arm; wherein the fourth arm region of the second single-stranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the seventh arm region of the fourth singlestranded DNA oligonucleotide to form a third double stranded region of a third nanostar arm, and ii) a sequence that is non-complementary to the seventh arm region that forms a third single-stranded overhang region of the third nanostar arm; and wherein the sixth arm region of the third single-stranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the eighth arm region of the fourth singlestranded DNA oligonucleotide to form a fourth double stranded region of a fourth nanostar arm, and ii) a sequence that is non-complementary to the eighth arm region that forms a fourth single-stranded overhang region of the fourth nanostar arm.13. The composition of aspect 9, wherein the nanostar condensate comprises a 5-arm nanostar comprising 5 different single stranded DNA oligonucleotides.14. The composition of aspect 9, wherein the nanostar condensate comprises a 6-arm nanostar comprising 6 different single stranded DNA oligonucleotides.15. The composition of any one of aspects 1 -14, wherein the nanostar condensate encapsulated by the plurality of nucleic acid surfactant molecules does not adhere to a hydrophobic surface.16. The composition of any one of aspects 1 -15, further comprising a payload.17. The composition of aspect 16, wherein the payload comprises a therapeutic agent, a gene therapy agent, a gene editing agent, or a differentiation agent.18. The composition of aspect 16 or 17, wherein the payload comprises a protein, a nucleic acid, a drug, an antisense oligonucleotide, an inhibitory RNA, a messenger RNA, a transgene, or an aptamer.19. The composition of aspect 18, wherein the protein is an antibody, a growth factor, a hormone, a neuropeptide, or an enzyme.20. The composition of aspect 18, wherein the inhibitory RNA is a microRNA (miRNA), short interfering RNA (siRNA), or short hairpin RNA (shRNA).21. The composition of aspect 17, wherein the gene editing agent is a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas endonuclease, a peptide-nucleic acid (PNA), a donor polynucleotide that can be used for homology-directed repair, a guide RNA, or a ribonucleoprotein comprising a Cas endonuclease complexed with a guide RNA.22. The composition of aspect 21 , wherein the guide RNA targets the Cas endonuclease to a target gene of interest for gene editing.23. The composition of any one of aspects 16-22, wherein the payload is covalently linked to the plurality of nucleic acid surfactant molecules or the plurality of DNA nanostars.24. The composition of any one of aspects 16-22, wherein the payload is conjugated to a single stranded nucleic acid, wherein the single stranded nucleic acid comprises a region that is complementary to and hybridizes to a single stranded region of each nucleic acid surfactantmolecule, thereby presenting the payload on the plurality of nucleic acid surfactant molecules encapsulating the nanostar condensate.25. The composition of aspect 24, wherein the payload is conjugated to a single stranded nucleic acid comprising a homopolymeric sequence.26. The composition of any one of aspects 16-22, wherein the payload is encapsulated by the nanostar condensate.27. The composition of any one of aspects 1 -26, further comprising a plurality of targeting agents conjugated to the plurality of nucleic acid surfactant molecules.28. The composition of aspect 27, wherein the targeting agents are antibodies, peptides, peptoids, or aptamers that specifically bind to a cell surface marker or a receptor on a target cell.29. The composition of any one of aspects 1-28, wherein the nucleic acid surfactant molecule is conjugated to a polymer.30. The composition of aspect 29, wherein the polymer is biodegradable.31 . The composition of aspect 29 or 30, wherein the polymer is selected from the group consisting of polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA), poly(e- caprolactone) (POL), poly(lactide-co-glycolide) (PLGA), and a copolymer of poly(lactide-co- glycolide) and poly(e-caprolactone) (PLGA / PCL).32. The composition of any one of aspects 1 -31 , further comprising a shell comprising a plurality of non-nucleic acid molecules.33. The composition of aspect 32, wherein the non-nucleic acid molecules are targeting agents or conjugated to targeting agents.34. The composition of aspect 32 or 33, wherein the non-nucleic acid molecules are selected from the group consisting of peptides, peptoids, peptide nucleic acids, aptamers, antibodies, and polymers.35. The composition of aspect 34, wherein the polymers are selected from the group consisting of polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA), poly(e- caprolactone) (POL), poly(lactide-co-glycolide) (PLGA), and a copolymer of poly(lactide-co- glycolide) and poly(e-caprolactone) (PLGA / PCL).36. The composition of any one of aspects 1 -35, further comprising a pharmaceutically acceptable excipient.37. A method of delivering a payload into a cell, the method comprising introducing the composition of any one of aspects 16-36 into the cell.38. The method of aspect 37, wherein the cell is an adult cell, a stem cell, or a progenitor cell.39. The method of aspect 37 or 38, wherein the delivery is performed in vitro, ex vivo, or in vivo.40. The method of any one of aspects 37-39 wherein the payload comprises a therapeutic agent, a gene therapy agent, a gene editing agent, or a differentiation agent.41 . The method of any one of aspects 37-40, wherein the payload comprises a protein, a nucleic acid, a drug, an antisense oligonucleotide, an inhibitory RNA, a messenger RNA, a transgene, or an aptamer.42. The method of aspect 41 , wherein the protein is an antibody, a growth factor, a hormone, a neuropeptide, or an enzyme.43. The method of aspect 41 , wherein the inhibitory RNA is a microRNA (miRNA), short interfering RNA (siRNA), or short hairpin RNA (shRNA).44. The method of aspect 40, wherein the gene editing agent is a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas endonuclease, a peptidenucleic acid (PNA), a donor polynucleotide that can be used for homology-directed repair, a guide RNA, or a ribonucleoprotein comprising a Cas endonuclease complexed with a guide RNA.45. The method of aspect 44, wherein the guide RNA targets the Cas endonuclease to a target gene of interest for gene editing.46. A method of decreasing size of the nanostar condensate, the method comprising increasing concentration of the nucleic acid surfactant molecule.47. A method of delivering a therapeutic agent to a subject, the method comprising administering the composition of aspect 17 to the subject.48. The method of aspect 47, wherein the therapeutic agent is a drug, protein, a nucleic acid, or an aptamer.49. The method of aspect 48, wherein the protein is an antibody, a growth factor, a hormone, a neuropeptide, or an enzyme.50. The method of aspect 48, wherein the nucleic acid is an antisense oligonucleotide, an inhibitory RNA, a transgene, a messenger RNA, a guide RNA, or a donor polynucleotide.51 . The method of aspect 50, wherein the inhibitory RNA is a microRNA (miRNA), short interfering RNA (siRNA), or short hairpin RNA (shRNA).52. A method of genetically modifying a cell, the method comprising introducing the composition of aspect 21 into the cell.EXPERIMENTAL

[0094] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are notintended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0095] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0096] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims.Example 1Nanostar Condensate Size Control with DNA Surfactant

[0097] Biomolecular condensates have a critical role in cellular function. As a tool to study properties of biomolecular condensates, synthetic biomolecules are widely used. The study of synthetic condensates introduces new design in biochemical industry and generates synthetic cells that helps understand roles of membraneless organelles in cellular functioning. In particular, the ability to control biomolecular synthetic condensates would enhance such condensates as common bioreactors and prompts for new drug delivery methods. However, the studies relevant to condensate size regulation are generally needed.

[0098] Here, we use the synthetic biomolecular model system, termed nanostar (NS). Nanostars are composed of single-stranded DNA (ssDNA) sequences that are designed such that they can self- assemble into certain-valence nanoparticles. Nanostars bind to each other at their palindromicssDNA sticky end. Below a critical temperature relevant to palindrome’s hybridization energy, nanostars phase separate and form a dynamic, disordered mesh, called nanostar droplets.

[0099] Inspired from the principles used in emulsion stabilization and dispersion polymerization, we design long stranded DNA that can control nanostar droplets. Previous study by Nguyen et al.1indicates that these long-stranded DNA, called DNA surfactants, localize to nanostar droplet interface and exhibit a surfactant- 1 ike behavior. They form a coating around nanostar droplets because DNA surfactants hybridize with individual nanostar but are excluded from the droplets due to the high entropic cost of confining long DNA strands inside the dense nanostar mesh. Such coating is designed to function like stabilizers in dispersion polymerization that inhibits condensate coalescing and adhesion, which subsequently limits droplet size.

[0100] Here, we examine the effect of long DNA surfactants on the size and adhesive properties of nanostar droplets. We find out that as we increase the DNA surfactant to Nanostar ratio, the mean size of droplets decreases. And as we further increase DNA surfactant concentration to Nanostar concentration, we generate sub-micron-scale micelle-like structures. Along with decrease in size, nanostar droplets coated with DNA surfactant also resist adhering to hydrophobic surfaces relative to only nanostar droplets, indicating sufficient surface coverage to protect unbounded ss-DNA parts of nanostars in the bulk. We also observe similar non- sticking behavior of nanostar droplets with DNA surfactant coverage on protein coated surfaces, depicted by increased droplet mobility on the surface, which proposes a potential drug delivery method.Results and DiscussionNanostar and DNA Surfactant Design and Assembly

[0101] The 4-arm Nanostars are assembled from four different ssDNA oligos, with the design followed from work established by Biffi et al. Each oligo is designed with a non-binding center section that separates the two sides capable of base-pair binding with two different oligos. The product, when fully annealed with each oligo binding with the other two oligos, will form an x-shape structure with four arms extending from the same shared non-binding core, called Nanostars (NS) (FIG. 1). At the end of each extending arm, there are 7 bases of ssDNA left unbounded. The end 6 bases are palindromic sticky end that enable NS-NS binding interaction. Whereas the middle one free nucleotide ensures flexibility between the binding sites and nanostar arms. NS-NS binding is activated upon addition of 1 M NaCI that screens the DNA backbone repulsion and is stabilized at a temperature lower than 36 G. The interconnected Nanostars form a transient network that phaseseparate from bulk phase, and together with the free nucleotides, gives nanostar condensate a liquidlike property.

[0102] Using auto-sticky Polymerase Chain Reaction (AS-PCR) technique, the DNA surfactants are polymerized with sticky ends that can by base-pairing bind with nanostars. The sticky ends on DNA surfactants are programmed complimentary to the whole Nanostar overhang sequence, with a total NS-surfactants hybridization energy AG = -12.7 kcal / mol under 1 M NaCI -. Under the same solution condition, the NS-NS or surfactant-surfactant hybridization energy, without the two extra base stacking energy around the unfounded free nucleotide, is AG = -7.2 kcal / mol, making them less favorable than NS-surfactants binding. (FIG. 1 ) The length of the double-stranded part in DNA surfactants is 405 bp, equivalently 135 nm, which is around 15 times larger than the previous estimation of Nanostar characteristic mesh grid size 9 nm.1Under the picture of worm-like-chain model of DNA double strand, the extended length brings high entropy cost of restricting long doublestranded DNA within droplet mesh grid.

[0103] Since DNA surfactants bind to nanostars but are entropically frustrated from the interconnecting nanostar mesh grid inside nanostar condensates, they mainly localize to the interface between NS-dilute and NS-dense phase. To visualize surfactant adhesion on NS droplet surface, we mix 4 / M nanostar and 0.4 M of DNA surfactant in 1 M NaCI. The sample is imaged under confocal microscope using droplet formation protocol. All the DNA surfactant molecules are fluorescently tagged with Cy3 fluorophore on the DNA phosphate backbone between overhand and double-stranded DNA (dsDNA) part. 5% of the nanostars are tagged with Cy5 fluorophore at similar location on their first arm. We observed a ring of higher fluorescence intensity on the surface of nanostar droplets under confocal microscope, which indicates a higher DNA surfactant concentration.1To analyze the concentration of DNA surfactant across the droplet interface, we plot fluorescence intensity radially from the center of nanostar droplet. We see a lower intensity within the droplet that indicates depletion of DNA surfactant in NS-dense phase. And at the droplet interface, we see a high fluorescence ring of width around M due to the diffraction limit of confocal microscope. The high fluorescence ring suggests that the DNA surfactant surrounds nanostar droplets, which is consistent with previous observations.DNA surfactant control nanostar droplet size.

[0104] As the DNA surfactant forms a shell surrounding an encapsulated nanostar droplet, we posited it could act as a buffer that inhibits droplet coalescence. And an increase in DNA surfactant concentration in nanostar condensates would decrease the critical volume of nanostar droplets whenDNA surfactant shell forms, which in return would result in a smaller nanostar droplet size. To examine this effect, we mix 2 / A / l DNA nanostar with a surfactant concentration varying from 0 juM to 0.6 M. Under 1 M NaCI salt condition, the mixture is initially brought up to 50 O for 30 minutes to reach homogeneous state. The sample is later fixed on a rotor at room temperature for droplet formation. The rotor prevents gravitational induced aggregation by constantly changing the sample orientation. The droplets are later transferred into the flow cell and left unperturbed for 30 minutes to let droplets fully sediment. Images are taken at a focal plane few microns above the bottom of the flow cells so that all sedimented droplets are captured. Since the height of flow cell is on the scale of 50 M, the droplet size increase due to gravity can be ignored. 5% of nanostars are Cy3 fluorescently tagged and the images are taken under fluorescent microscopy for more accurate droplet radius measurements even when the microscope focal plane does not align with droplet center plane.

[0105] We observe a decrease in nanostar condensate gize with increasing DNA surfactant concentration. (FIG. 2) The droplet sizes data are measured from images using computer program based on the algorithm developed by Crocker and Grier -. The algorithm generated size data are compared with droplet size on picture to make sure that the generated size error is within 10% measured radius. In the size distribution plot, we discard droplets data with radius smaller than one pixel (0.18 mum) since they are noise sensitive. The size distribution is presented in complimentary cumulative distribution function (CCDF) defined as CCDF = 1 - *f(x' dx' to reduce noise and prevent binning effect in histogram.

[0106] For each sample, the droplet size distribution can be coarsely approximated as exponential decay function (FIG. 2). The exponential decay of droplet count with increasing droplet volume, we propose, is from stochastic coalescing between the nanostar droplets. As the samples are directly transformed to room temperature, they undergo a fast nucleation phase. When the nucleation sites coalesce and ripen, the DNA surfactant in a droplet increases to the order of r3and is expelled to the surface. However, as the surface area increases to the order of r2, the DNA surfactant concentration would increase on the surface and eventually form a protective shell, by which the droplets can only grow from coalescing. Our shape of CCDF is in accordance with observations and simulations by Lee et al.5, where the exponential size distribution also appear for condensates undergo fast nucleation followed by coalescence.High Surfactant to NS ratio generates sub-micron-scale micelle-like structure.

[0107] In FIG. 2, we observe that when surfactant to NS ratio increases to 1 :3, the nanostar droplets become difficult for optical microscope to resolve. And if we further increase the surfactant to NS ratio to 1 :1 , with each kind at a concentration of 2 M, we observe a homogeneous background, which indicates that the structure formed are smaller than the optical microscope resolution limit.

[0108] To determine the size of submicron-scale assembled structure at high surfactant to NS ratio, we use dynamic light scattering (DLS) technique. For our measurements, the size distribution is reconstructed from those diffusion coefficients using globular protein. The intensity distribution (FIG. 3A) indicates percentage of light scattered with certain solute radius. From the plot, we see that some dispersants in NS-surfactant mixture have size larger than either single nanostar or DNA surfactant. We conclude that the larger parts are the assembled micelles at high surfactant to NS ratio.

[0109] The correlation function in FIG. 3A also clearly illustrates that the mixture of NS and DNA surfactant contains particles that diffuse slower than individual nanostar or DNA surfactant particle, which indicates presence of larger particles. However, the intensity distribution function is only an approximation fitted from correlation function based on the assumption that the particle distribution is in Gaussian shape. For instance, in the nanostar only sample, the Gaussian fit that centers around 102 nm radius data, we conclude, is simply a coarse fitting of all the large particle noise in the correlation function. Moreover, the particle radius is not applicable for only nanostar and only DNA surfactant measurements due to their non-spherical shape. Nevertheless, the data provides sufficient evidence for the existence of sub-micron- sized particles in solution at high surfactant-NS ratio.

[0110] Due to the indirect nature of light scattering technique, we also use transmission electron microscopy (TEM) to directly confirm the existence of assembled structure. To prevent aggregation during the flash dry process prior to TEM imaging, the sample is diluted down 5 times, with 0.4 / M NS and 0.4 M DNA surfactant. Despite the sample concentration change, surfactant to NS ratio is maintained, which should preserve the coalescing behaviors of nanostar droplets. Therefore, the obtained image can still represent the size of nanostar condensates at high surfactant to NS ratio to some degree. As in FIG. 3B, TEM reveals roughly the droplet size expected. The droplets are imaged as dark blobs with long stranded DNA visible as wiggling lines in the right picture. The droplet size might be slightly inflated since the sample is flash dried on the plasma cleaned surface, where the spherical droplets flatten on the slide, which may result in a shade radius than the sphere radius.DNA surfactants modulate the adhesive properties of nanostar droplets.

[0111] To examine the DNA surfactant coverage on nanostar droplets, we place the droplets on hydrophobic surfaces. Since ssDNA are more likely to stuck on hydrophobic surfaces than dsDNA, the nanostar droplets tends to wet onto the hydrophobic surface, as previously demonstrated by Saleh et al.5However, for droplets coated with DNA surfactant, the surfactant would form a dsDNA shell that prevents the internal ssDNA from adhering to surface. After leaving the sample on surface for 30 minutes for nanostar droplets to sufficiently sediment and interact with hydrophobic glass surface, we take pictures every 30 seconds to determine the droplet movement. The image arrays FIG. 4 with 30 seconds interval show that most of the droplets within NS-surfactant mixture sample retain Brownian motion, whereas the pure nanostar droplets seem glued on the surface. This result indicates that DNA surfactant has sufficient coverage around the nanostar droplets to form a protective shell. Noticeably, we also observe a decrease in condensate size with addition of DNA surfactant as discussed previously.

[0112] Such enhanced mobility of nanostar droplets with DNA surfactant coating is also observed on other types of surfaces such as bovine serum albumin (BSA) protein coated glass surfaces. As indicated in the image arrays FIG. 4 with 15 minutes interval, a relatively small amount of DNA surfactant added in nanostar solution is capable of resisting relative large droplet from sticking, strengthening the point that the DNA surfactant can sufficiently prevent the nanostar inside from interacting with exterior medium.

[0113] Interestingly, the size decrease is hardly noticeable for the case of 2 M nanostar - 0.1 M DNA surfactant mixture in FIG. 4 and FIG. 2. This effect could arise from the fact that the DNA surfactant tends to saturate the nanostars in bulk phase before adhering to dense phase surface as they are less entropically constrained in the prior case. Previous research suggests that nanostar concentration is typically on the order of 0.01 / M at dilute phasez, which will resulting a thresholding concentration on the order of 0.04 M, for 4 arm nanostar full saturation, before DNA surfactants start actively controlling condensate size. Such result explains the reduced size control effect of 0.1 M DNA surfactant compared with other concentrations.Conclusions

[0114] We have shown a liquid-liquid phase separation (LLPS) system: NS-DNA surfactant system, that is capable to generate condensates with a tunable size ranging from a radius of 10 / M to 30 nm. We also demonstrated that the DNA surfactant forms a shell around nanostar condensates that prevents the droplets from adhering to hydrophobic and BSA coated surfaces. The size controland anti-adhesion property of the system suggests a new design for drug delivery device and provides insights for size control mechanism of biomolecular condensates.

[0115] Future design direction would be to reduce the polydispersity of condensate formed as well as to decrease the DNA surfactant threshold concentration for droplet size control. The droplet can design to be more monodisperse by strengthen the DNA surfactant protective shell that prevents further coalescence. Use higher NS-surfactant hybridization energy or ligate them could be of potential options. The DNA surfactant threshold concentration can be decreased by decreasing the free nanostars in bulk solution. Use stronger NS-NS hybridization energy or lowering the annealing temperature could be of choice.MethodsAnnealing of nanostars.

[0116] Four flavors of ssDNA oligos are mixed with each flavor having a concentration of 50 / M and a total of 50 L. The mixture is initially heated up to 95 *C for 10 minutes and cooled to 4 C with a constant rate of 0.5 °C / min in thermocycler.Creating DNA surfactant.

[0117] DNA surfactants are created using PCR with Taq Polymerase and lambda phage DNA. The forward primers have an overhang that is separated from the rest lambda genome binding sequence by an empty nucleotide. Since the polymerization stops at the empty nucleotide, the PCR product is left with a short sequence of ssDNA at the forward primer end.BSA Protein coated slide preparation.

[0118] The cover slips are cleaned by emerging in 2% Hellmanex solution and sonicated for 10 minutes. They are later submerged in 10 mg / mL BSA protein solution for at least 24 hours. The BSA protein coating helps prevent nanostar condensates from adhering to the glass surface and maintain droplet in spherical shape that enhance image quantification. Finally, the cover slips are assembled into flow cells by sandwiching melted parafilm as a spacer in between.Hydrophobic surface slide preparation.

[0119] The cover slip surfaces are rinsed by Acetone, Isopropanol, water, and plasma cleaned. The surfaces are later treated by Sigmacote, a siliconizing reagent that forms a thin hydrophobiccoating on the glass surface, for 30 seconds. The reagents are later flushed away with ethanol and air dried to make flow cells.Droplet formation protocol.

[0120] The Nanostars and DNA surfactant are initially mixed in 1 M NaCI solution and heated up to 50 G for 30 minutes to unhybridize. Then, they are transformed to room temperature to phase separate. The samples are placed on a rotor with 5 rotations / min to prevent aggregation due to gravity. After one or two hours of rotation, the samples are transferred into flow cells.Dynamic Light Scattering.

[0121] DLS is carried out using Wyatt Dynamics machine, with correlation data analyzed using Wyatt Dynamics software. Each sample volume is transferred into cuvettes and measured twice. Each size distribution is calculated from averaging correlation function from a total of 20 trials with each trial correlated for 5 seconds.Fluorescent imaging.

[0122] The images with two channels color is taken through Leica. The images in droplet size section are taken from Ti2 (Eve) with magnification of 20x or 40x lenses, depending on the droplet size.TEM imaging.

[0123] The sample holder is initially plasma cleaned so that the DNA droplets would adhere to the surface. They are later dried and imaged.References

[0124] D. T. Nguyen, B.-j. Jeon, G. R. Abraham and O. A. Saleh, Langmuir, 2019, 35, 14849- 14854.

[0125] S. Biffi, R. Cerbino, F. Bomboi, E. M. Paraboschi, R. Asselta, F. Sciortino and T. Bellini, Proceedings of the National Academy of Sciences, 2013, 110, 15633-15637.

[0126] J. SantaLucia, Proceedings of the National Academy of Sciences, 1998, 95, 1460-1465.

[0127] J. C. Crocker and D. G. Grier, Journal of Colloid and Interface Science, 1996, 179, 298-310.

[0128] D. S. W. Lee, C.-H. Choi, D. W. Sanders, L. Beckers, J. A. Riback, C. P. Brangwynne and N. S. Wingreen, Nature Physics, 2023, 19, 586-596.

[0129] O. A. Saleh, B. jin Jeon and T. Liedl, Proceedings of the National Academy of Sciences, 2020, 117, 16160-16166.

[0130] N. Conrad, G. Chang, D. K. Fygenson and O. A. Saleh, The Journal of Chemical Physics, 2022, 157, 234203.

Claims

What is claimed is:1 . A composition comprising:(a) a nanostar condensate comprising a plurality of DNA nanostars assembled from a plurality of single-stranded DNA oligonucleotides, wherein each DNA nanostar comprises: a core and a plurality of arms extending from the core, wherein each arm comprises i) a double-stranded region and ii) a single-stranded overhang region comprising a binding site for the single-stranded overhang region of an arm of another DNA nanostar, wherein the plurality of DNA nanostars assemble into a mesh structure in the nanostar condensate; and(b) a plurality of nucleic acid surfactant molecules, wherein each nucleic acid surfactant molecule comprises a double-stranded region and a single-stranded overhang region, wherein the single stranded overhang region of the nucleic acid surfactant molecule is complementary to and hybridizes to the single-stranded overhang regions of the plurality of arms, wherein the plurality of nucleic acid surfactant molecules encapsulate the nanostar condensate.

2. The composition of claim 1 , wherein the binding site comprises a palindromic sequence.

3. The composition of claim 2, wherein the palindromic sequence is 6 nucleotides to 12 nucleotides in length.

4. The composition of any one of claims 1 -3, wherein each arm further comprises a linker comprising one or more unpaired bases between the double-stranded region and the binding site.

5. The composition of claim 4, wherein the linker is one to three nucleotides in length.

6. The composition of any one of claims 1 -5, wherein the nucleic acid surfactant comprises a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).

7. The composition of any one of claims 1 -6, wherein the nanostar condensate has a radius having a length in a range from about 30 nm to about 10 pm.

8. The composition of any one of claims 1-7, wherein each DNA nanostar comprises 3 arms, 4 arms, 5 arms, or six arms.

9. The composition of any one of claims 1-8, wherein the plurality of single-stranded DNA oligonucleotides comprises 3 to 6 different single-stranded DNA oligonucleotides.

10. The composition of claim 9, wherein the nanostar condensate comprises a 3-arm nanostar comprising 3 different single stranded DNA oligonucleotides.11 . The composition of claim 9, wherein the nanostar condensate comprises a 4-arm nanostar comprising 4 different single stranded DNA oligonucleotides.

12. The composition of claim 11 , wherein each DNA nanostar is assembled from a plurality of single-stranded DNA oligonucleotides, wherein the plurality of single-stranded DNA oligonucleotides comprises: a first single-stranded DNA oligonucleotide comprising a first core region, wherein the first core region is located between a first arm region and a second arm region; a second single-stranded DNA oligonucleotide comprising a second core region, wherein the second core region is located between a third arm region and a fourth arm region; a third single-stranded DNA oligonucleotide comprising a third core region, wherein the third core region is located between a fifth arm region and a sixth arm region; and a fourth single-stranded DNA oligonucleotide comprising a fourth core region, wherein the fourth core region is located between a seventh arm region and a eighth arm region; wherein the first arm region of the first single-stranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the third arm region of the second singlestranded DNA oligonucleotide to form a first double stranded region of a first nanostar arm, and ii) a sequence that is non-complementary to the third arm region that forms a first single-stranded overhang region of the first nanostar arm; wherein the second arm region of the first single-stranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the fifth arm region of the third single-stranded DNA oligonucleotide to form a second double stranded region of a second nanostar arm, and ii) a sequence that is non-complementary to the fifth arm region that forms a second single-stranded overhang region of the second nanostar arm;wherein the fourth arm region of the second single-stranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the seventh arm region of the fourth singlestranded DNA oligonucleotide to form a third double stranded region of a third nanostar arm, and ii) a sequence that is non-complementary to the seventh arm region that forms a third single-stranded overhang region of the third nanostar arm; and wherein the sixth arm region of the third single-stranded DNA oligonucleotide comprises i) a sequence that is complementary to and hybridizes to the eighth arm region of the fourth singlestranded DNA oligonucleotide to form a fourth double stranded region of a fourth nanostar arm, and ii) a sequence that is non-complementary to the eighth arm region that forms a fourth single-stranded overhang region of the fourth nanostar arm.

13. The composition of claim 9, wherein the nanostar condensate comprises a 5-arm nanostar comprising 5 different single stranded DNA oligonucleotides.

14. The composition of claim 9, wherein the nanostar condensate comprises a 6-arm nanostar comprising 6 different single stranded DNA oligonucleotides.

15. The composition of any one of claims 1 -14, wherein the nanostar condensate encapsulated by the plurality of nucleic acid surfactant molecules does not adhere to a hydrophobic surface.

16. The composition of any one of claims 1-15, further comprising a payload.

17. The composition of claim 16, wherein the payload comprises a therapeutic agent, a gene therapy agent, a gene editing agent, or a differentiation agent.

18. The composition of claim 16 or 17, wherein the payload comprises a protein, a nucleic acid, a drug, an antisense oligonucleotide, an inhibitory RNA, a messenger RNA, a transgene, or an aptamer.

19. The composition of claim 18, wherein the protein is an antibody, a growth factor, a hormone, a neuropeptide, or an enzyme.

20. The composition of claim 18, wherein the inhibitory RNA is a microRNA (miRNA), short interfering RNA (siRNA), or short hairpin RNA (shRNA).21 . The composition of claim 17, wherein the gene editing agent is a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas endonuclease, a peptidenucleic acid (PNA), a donor polynucleotide that can be used for homology-directed repair, a guide RNA, or a ribonucleoprotein comprising a Cas endonuclease complexed with a guide RNA.

22. The composition of claim 21 , wherein the guide RNA targets the Cas endonuclease to a target gene of interest for gene editing.

23. The composition of any one of claims 16-22, wherein the payload is covalently linked to the plurality of nucleic acid surfactant molecules or the plurality of DNA nanostars.

24. The composition of any one of claims 16-22, wherein the payload is conjugated to a single stranded nucleic acid, wherein the single stranded nucleic acid comprises a region that is complementary to and hybridizes to a single stranded region of each nucleic acid surfactant molecule, thereby presenting the payload on the plurality of nucleic acid surfactant molecules encapsulating the nanostar condensate.

25. The composition of claim 24, wherein the payload is conjugated to a single stranded nucleic acid comprising a homopolymeric sequence.

26. The composition of any one of claims 16-22, wherein the payload is encapsulated by the nanostar condensate.

27. The composition of any one of claims 1-26, further comprising a plurality of targeting agents conjugated to the plurality of nucleic acid surfactant molecules.

28. The composition of claim 27, wherein the targeting agents are antibodies, peptides, peptoids, or aptamers that specifically bind to a cell surface marker or a receptor on a target cell.

29. The composition of any one of claims 1 -28, wherein the nucleic acid surfactant molecule is conjugated to a polymer.

30. The composition of claim 29, wherein the polymer is biodegradable.31 . The composition of claim 29 or 30, wherein the polymer is selected from the group consisting of polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA), poly(e- caprolactone) (PCL), poly(lactide-co-glycolide) (PLGA), and a copolymer of poly(lactide-co- glycolide) and poly(e-caprolactone) (PLGA / PCL).

32. The composition of any one of claims 1 -31 , further comprising a shell comprising a plurality of non-nucleic acid molecules.

33. The composition of claim 32, wherein the non-nucleic acid molecules are targeting agents or conjugated to targeting agents.

34. The composition of claim 32 or 33, wherein the non-nucleic acid molecules are selected from the group consisting of peptides, peptoids, peptide nucleic acids, aptamers, antibodies, and polymers.

35. The composition of claim 34, wherein the polymers are selected from the group consisting of polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA), poly(e- caprolactone) (PCL), poly(lactide-co-glycolide) (PLGA), and a copolymer of poly(lactide-co- glycolide) and poly(e-caprolactone) (PLGA / PCL).

36. The composition of any one of claims 1-35, further comprising a pharmaceutically acceptable excipient.

37. A method of delivering a payload into a cell, the method comprising introducing the composition of any one of claims 16-36 into the cell.

38. The method of claim 37, wherein the cell is an adult cell, a stem cell, or a progenitor cell.

39. The method of claim 37 or 38, wherein the delivery is performed in vitro, ex vivo, or in vivo.

40. The method of any one of claims 37-39 wherein the payload comprises a therapeutic agent, a gene therapy agent, a gene editing agent, or a differentiation agent.41 . The method of any one of claims 37-40, wherein the payload comprises a protein, a nucleic acid, a drug, an antisense oligonucleotide, an inhibitory RNA, a messenger RNA, a transgene, or an aptamer.

42. The method of claim 41 , wherein the protein is an antibody, a growth factor, a hormone, a neuropeptide, or an enzyme.

43. The method of claim 41 , wherein the inhibitory RNA is a microRNA (miRNA), short interfering RNA (siRNA), or short hairpin RNA (shRNA).

44. The method of claim 40, wherein the gene editing agent is a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a Cas endonuclease, a peptidenucleic acid (PNA), a donor polynucleotide that can be used for homology-directed repair, a guide RNA, or a ribonucleoprotein comprising a Cas endonuclease complexed with a guide RNA.

45. The method of claim 44, wherein the guide RNA targets the Cas endonuclease to a target gene of interest for gene editing.

46. A method of decreasing size of the nanostar condensate, the method comprising increasing concentration of the nucleic acid surfactant molecule.

47. A method of delivering a therapeutic agent to a subject, the method comprising administering the composition of claim 17 to the subject.

48. The method of claim 47, wherein the therapeutic agent is a drug, protein, a nucleic acid, or an aptamer.

49. The method of claim 48, wherein the protein is an antibody, a growth factor, a hormone, a neuropeptide, or an enzyme.

50. The method of claim 48, wherein the nucleic acid is an antisense oligonucleotide, an inhibitory RNA, a transgene, a messenger RNA, a guide RNA, or a donor polynucleotide.51 . The method of claim 50, wherein the inhibitory RNA is a microRNA (miRNA), short interfering RNA (siRNA), or short hairpin RNA (shRNA).

52. A method of genetically modifying a cell, the method comprising introducing the composition of claim 21 into the cell.

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