RNA-Based Coacervates as a Model for ... - ACS Publications

Subscriber access provided by Northern Illinois University

Article

RNA-based coacervates as a model for membraneless organelles: Formation, properties, and interfacial liposome assembly William M. Aumiller, Fatma Pir-Cakmak, Bradley W. Davis, and Christine D. Keating Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02499 • Publication Date (Web): 06 Sep 2016 Downloaded from http://pubs.acs.org on September 8, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

RNA-based coacervates as a model for membraneless organelles: Formation, properties, and interfacial liposome assembly William M. Aumiller, Jr.†, Fatma Pir Cakmak, Bradley W. Davis‡, and Christine D. Keating* Department of Chemistry, Pennsylvania State University, University Park, PA 16802, United States

ABSTRACT Liquid-liquid phase separation is responsible for formation of P granules, nucleoli, and other membraneless subcellular organelles composed of RNA and proteins. Efforts to understand the physical basis of liquid organelle formation have thus far focused on intrinsically disordered proteins (IDPs) as major components that dictate occurrence and properties. Here, we show that complex coacervates composed of low complexity RNA (polyuridylic acid, polyU) and short polyamines (spermine and spermidine) share many features of IDP-based coacervates. PolyU/polyamine coacervates compartmentalize biomolecules (peptides, oligonucleotides) in a sequence- and length- dependent manner. These solutes retain mobility within the coacervate droplets, as demonstrated by rapid recovery from photobleaching. Coacervation is reversible with changes in solution temperature due to changes in the polyU structure that impact its interactions with polyamines. We further demonstrate that lipid vesicles assemble at the droplet interface without impeding RNA entry/egress. These vesicles remain intact at the interface and can be released upon temperature-induced droplet dissolution.

ACS Paragon Plus Environment

1

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 2 of 40

KEYWORDS: diffusion, compartmentalization, temperature-dependent, lipid vesicle, liquid organelle

INTRODUCTION A number of cytoplasmic and nucleoplasmic non-membrane bound organelles comprised of RNA and protein, such as nucleoli1 and P granules,2 have been reported to have liquid phase characteristics.3-11 An increasing number of other non-membrane bound organelles are candidates for liquid phase separation, including Cajal bodies, nuclear speckles, paraspeckles, and PML bodies in the nucleoplasm and stress granules and germ granules in the cytoplasm.12-13 Liquid-liquid phase separation (LLPS), thought to be the mechanism of intracellular droplet formation, is common in macromolecule-containing aqueous solutions, and researchers have developed experimental model systems based on these solutions in order to understand aspects of intracellular liquid organelles, such as what dictates their molecular occupancy and formation/disassembly.14-17 Complex coacervation, a class of LLPS that occurs when two oppositely charged polyelectrolytes undergo electrostatic attraction to form a dense, polyelectrolyte rich phase (the coacervate phase) and a much larger dilute phase, is an appealing model for intracellular phase separation. It can occur with pairs of synthetic polymers or biomacromolecules, such as RNA and short cationic peptides,15 nucleotides and cationic peptides,18-19 or two oppositely-charged peptides.20 Coacervate phases can be dissolved by changes in solution conditions, such as increasing the salt concentration,21-22 changing the pH,19 changing the overall charge of the polyelectrolyte (i.e. phosphorylation)15 and in some cases changing the temperature.23 These stimuli can also control the formation/disassembly of


2

Page 3 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

membraneless organelles and their major protein components,3,

8, 24

which makes complex

coacervation an excellent mimic for these structures. Key protein components of many membraneless organelles are characterized by intrinsically disordered regions (IDRs), sometimes containing low complexity sequences (LCSs).25-26 These regions lack a well-defined structure and are more flexible as compared to globular folds. They are often composed of repeats of charged amino acids (both positive and negative) – which makes them similar to polyelectrolytes that participate in coacervation. Indeed, these sequences that make the proteins amenable to undergoing phase separation are thought to be a driving force for phase separation in vivo.25-26 Membraneless organelles are often dynamic in nature, responding to environmental cues. For example, intracellular droplets composed largely of a construct of the protein Ddx4, rapidly respond to temperature changes.3 Ddx4 is found in germ granules and nuage and contains an intrinsically disordered N terminus. In vitro Ddx4 droplet behavior was dependent on solution conditions: decreasing the temperature promoted phase separation.3 Thermally-activated phase separation could be important in vivo, for example in driving formation of stress granules. Exposing HeLa cells expressing a fused in sarcoma – green fluorescent protein (FUS-GFP) fusion protein to heat stress caused formation of stress granules rich in the FUS-GFP.27 This is significant because FUS has been implicated in DNA repair. The opposite thermal responses of these two protein-based systems suggests that Ddx4 exhibits upper critical solution temperature (UCST) behavior, while FUS exhibits lower critical solution temperature (LCST) behavior. Differences in peptide sequence have been shown to lead to both types of behavior in single-peptide systems.28 While the importance of protein sequence and structure has been and continues to be studied, less attention has been paid to the role of RNA in forming membraneless organelles.


3

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 4 of 40

Membraneless organelles are important in RNA processing, and RNA recognition motifs and/or multivalent binding sites are common in proteins associated with membraneless organelles.11 The presence of RNA promotes phase separation in many cases.5, 8-9, 11 Like proteins, RNA can serve as a seed for formation of membraneless organelles.29 Long noncoding RNA (lncRNA)30-31 sequences range anywhere from 200 bases to tens of kilobases and often contain repetitive sequences.32 For example, a lncRNA known as NEAT1 has been shown to be a key structural component of the membraneless organelles known as paraspeckles, found in the nucleus.33 RNA binding proteins, which recognize particular RNA sequences, are clearly important in organelle formation, however we hypothesize that the more generally polyanionic nature of lncRNA could also contribute to a role in complex coacervation. Condensation of nucleic acids by polycations of various sizes, ranging from small polyamines (spermine, spermidine, cobalthexamine) to larger polyelectrolytes (polylysine and histone proteins) has been studied extensively,34-35 but not generally in the context of LLPS. Complex coacervation has been observed in nucleic acid-cation systems, where coacervation of plasmid DNA or small interfering RNA is of considerable interest for drug delivery.36-39 We chose as our polycations the small naturally-occurring polyamines, spermine and spermidine, which have numerous intracellular functions, such as maintaining macromolecular structure and stability and regulation of transcription and translation, among many others.40 Intracellularly, polyamines are present at millimolar concentrations and a majority fraction are found as DNAor RNA-polyamine complexes.41 The liquid-crystalline behavior of DNA/spermine and DNA/spermidine complexes has been reported previously.42 Here, we investigated coacervates formed in the absence of proteins or peptides by combining long, low complexity RNA (polyuridylic acid, polyU) and polyamines (spermine and


4

Page 5 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

spermidine) in order to understand key factors that determine phase separation and molecular occupancy of the resulting droplets. We found that the phase separation of polyU/polyamines was temperature dependent – the coacervates formed only above ambient temperature (~ 21oC). This was caused by changes in the structure of the polyU RNA as a function of temperature. The RNA/polyamine liquid droplets compartmentalized both peptides and short, single-stranded RNAs to different degrees based on their sequence and length. RNA oligonucleotides remained mobile within the polyU/spermine droplets, as determined by rapid recovery after photobleaching. Finally, we showed that liposomes (~90 nm diameter) assembled at the interface of the coacervate droplets with the surrounding solution. This interfacial layer of liposomes did not impede entry/egress of RNA nor cooling-induced dissolution of the droplets, and the individual liposomes remained intact after release by droplet dissolution.

RESULTS AND DISCUSSION Formation of polyU/spermine and polyU/spermidine coacervates We first determined what solution conditions would lead to phase separation in these experimental model systems, followed by characterization of the resultant coacervates. Spermine tetrahydrochloride or spermidine trihydrochloride were added to polyU RNA, (MW = ~600-1000 kDa) at low ionic strength (5 mM HEPES, pH 7.6, 1 mM MgCl2) at 37 oC to form turbid solutions (Scheme 1 and Figure 1A). Inspection of the turbid solutions by optical microscopy revealed the presence of spherical liquid coacervate droplets (Figures 1B and 1C). Centrifugation of the bulk samples resulted in a very small, coalesced coacervate phase at the bottom of the centrifuge tube. Both coacervate samples had polydisperse droplet sizes, but the spermine coacervates were larger, on average (polyU/spermine average diameter: 3 ± 2 µm,


5

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 6 of 40

polyU/spermidine coacervates: 1.8 ± 0.8 µm). Figure 1D shows solution turbidity as a function of polyamine concentration with a fixed concentration of polyU. For each sample, we estimated the ratio of positively to negatively charged groups at pH 7.6, based on polyelectrolyte concentration and pKa. Spermine formed coacervates with polyU RNA much more readily than spermidine. Spermine/PolyU coacervates formed at a critical coacervate concentration (CCC), or minimum concentration necessary for coacervate formation, of ~180 µM spermine, which corresponds to a 0.7 [+]/[-] charge ratio. An order of magnitude more spermidine was required to form coacervates (CCC = 9 [+]/[-] charge ratio or ~2.9 mM spermidine). Total charge per molecule (3.7 charges per molecule for spermine and 2.9 for spermidine at pH 7.6) rather than charge density (0.018 for spermine and 0.020 for spermidine, expressed as positive charge per molecular weight) determined coacervation behavior in these systems. This can be understood in terms of greater multivalency of interaction between the spermine and RNA as compared to the spermidine and RNA.15, 43 These findings are consistent with differences in binding affinities and precipitation conditions between spermine and spermidine with DNAs and RNAs that have been reported previously.44-47 The interaction between these polyamines and nucleic acids is thought to be largely charge-based at low ionic strength but also can involve additional, more sequencedependent interactions such as hydrogen bonding with the nucleobases.46-50 There was an increase and subsequent decrease in turbidity with increasing spermidine at the charge ratios tested. Spermine also showed a slight drop in turbidity at the highest ratio tested. This behavior has been observed with spermine and spermidine in complexation with DNA.42 Other coacervate systems in which the polyelectrolytes are more similar in molecular weight have a turbidity maximum when the charge ratios are matched.22-23 In our case, excess polycation (per mole in the case of spermine, per mole and charge in the case of spermidine) was


6

Page 7 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

needed to condense the much longer polyU. This is similar to other coacervate systems in which one of the components has low multivalency compared to the other.18-19,

51

Many coacervates

typically reach a turbidity maximum, followed by turbidity loss while increasing one polyelectrolyte concentration while the other is fixed.22,

51

This can be explained as

“overcharging” of the coacervates as the polyelectrolyte concentration increases, and the soluble polyelectrolyte complexes that form are electrostatically repulsive.36, 52 The excess of polyamines resulted in formation of neutrally or nearly neutrally charged droplets. The zeta potential of the polyU/spermine droplets was +0.3 ± 5.8 mV, while the polyU/spermidine droplets was -5 ± 5 mV (measured at 0.5% poly U and 0.05% polyamine). Spermine/polyU coacervates were also much more salt resistant than spermidine/polyU coacervates, as seen in Figure 1E. Addition of just 25 mM NaCl caused spermidine/polyU coacervates to dissolve, while the spermine/polyU coacervates were stable up to 175 mM NaCl.


7

Langmuir

A H 3N

N H2

pKa 10.9

8.4

H2 N

NH 3

7.9

10.1

spermine H 3N

NH 3

N H2

pKa 10.9

8.4

9.9

spermidine

-

-

-

-

-

-

+ + + -

+

polyamine

+

-

+

poly U

+ +

- + + +- + - + + + + + - + + + + + + + + + + + - -

+

-

-

-

+ +

-

-

-

-

+

+

-

+ + - + + + + - + +- - - - -+ + + + + + - -

+ + -

+ +

-

+

B

+ +

+

+

+

+ +

+

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 8 of 40

coacervation

Scheme 1. (A) Chemical structures of the polyamines used with indicated pKa values. (B) Addition of the polyamine to PolyU in solution results in coacervation.


8

Page 9 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

Figure 1. Characterization of polyU/polyamine coacervates. (A) Photographs of bulk polyU/spermine coacervates as prepared (left) and after centrifugation (right). A dye-labeled RNA (Alexa Flour 647 poly A15) was added to aid in visualization. (B) A transmitted light differential interference contrast (DIC) image of polyU/spermine coacervates. The concentrations (in weight percent) of components were 0.05 % polyU and 0.5 % spermine (a charge ratio of 56:1). (C) DIC image of polyU/spermidine coacervates. The concentrations were 0.05 % polyU and 0.5 % spermidine (a charge ratio of 61:1). (D) Turbidity as a function of charge ratio for spermine (blue trace) and spermidine (red trace). Spermine coacervates required less charge for phase separation and had a higher turbidity than spermidine coacervates. PolyU concentration was fixed at 0.05%. (E) Effect of NaCl concentration on spermine (blue) and spermidine coacervates (red). Spermidine coacervates dissolved with just 25 mM NaCl, while spermine coacervates dissolved at 200 mM NaCl. The concentrations were 0.05 % polyU and 0.5 % spermine or spermidine. In panel (D) and (E) lines were drawn to guide the eye. All data were collected at 37 °C. Buffer conditions were 5 mM HEPES, pH 7.6 and 1 mM MgCl2. Temperature dependence of PolyU/spermine and polyU/spermidine coacervation. We also investigated the temperature dependence of coacervation in these systems. At typical laboratory temperature (~18 oC) we observed a turbid solution initially upon mixing, but


9

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 10 of 40

solutions became transparent after approximately 10 minutes. However, an increase in temperature of the solution from holding the centrifuge tube in hand caused turbidity again. We investigated this temperature-dependent behavior quantitatively by measuring sample turbidity and absorbance as a function of temperature for polyU/spermine, polyU/spermidine, and polyU alone (Figure 2). The cloud points were the same for both the polyU/spermine and polyU/spermidine systems, with little evidence of hysteresis (cloud points for heating and cooling were: 21 and 22 oC, respectively). Derivative plots (Figure 2A and B, bottom panels, dA/dT vs temperature) highlight the steepness of the phase transition. Although heating to the cloud point was necessary to observe turbidity, further heating the coacervates beyond the cloud point temperature led to decreased turbidity. This could be due to changes in droplet number and/or size, perhaps due to sedimentation or, as has been proposed for other complex coacervate systems, could indicate weakening interactions between the polyelectrolytes.23, 53 A solution of polyU with no polyamines did not become turbid (black traces) indicating that polyU alone cannot undergo phase separation under these conditions. We also monitored the disassembly of the coacervates with decreasing temperature using transmitted light microscopy (Figure 2C). The coacervate droplets shrank with time, eventually dissolving to form a one-phase solution.


10

Page 11 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

Figure 2. Effect of temperature on coacervation of polyU and polyamines. For (A) and (B) panels, black points are polyU alone, blue points are polyU/spermine, and red points are polyU/spermidine. Lines are drawn to guide the eye. The time elapsed between each temperature measurement was 5 minutes. (A). Turbidity (top), Absorbance (middle) and Derivative (bottom) plots measured at 500 nm with increasing temperature. PolyU/spermine coacervates become more turbid than spermidine, but the turbidity decreased more than spermidine. The cloud point


11

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 12 of 40

is nearly the same for both spermine and spermidine. PolyU alone does not undergo phase separation. (B) Turbidity (top), Absorbance (middle) and Derivative plots (bottom) with decreasing temperature. Spermine and spermidine coacervates had a similar turbidity and absorbance until the cloud point was reached. (C) Images of polyU/spermine coacervate droplet disassociation. The solutions were incubated at 37 oC for 30 minutes to allow coacervate droplets to settle to the coverslip surface. At time = 0, the temperature of the stage was decreased at a rate of 10 oC per minute to 10 oC. By 5 minutes, droplets are nearly dissolved; they are completely dissolved at 10 minutes. For (A), (B), and (C), the concentrations were 0.05 % polyU and 0.5 % spermine (a charge ratio of 56:1) or 0.5 % spermidine (a charge ratio of 61:1).

. To further investigate this striking temperature dependence, we checked for changes in RNA secondary structure by monitoring the absorbance at 260 nm (A260) as a function of temperature (Figures 3, top panel and S1). At low temperature, from 1 oC to 12 oC, the absorbance steadily increased, and subsequently plateaued above 12 oC. This is a trend very similar with what others have observed for polyU under other buffer and salt conditions.54-56 This hyperchromicity of polyU is caused by the gradual loss of secondary structure with increasing temperature, which is analogous to an increase in absorbance from melting double stranded DNA and RNA. Circular dichroism and other methods provide further evidence for polyU structure at low temperatures.55 This structure has been proposed to arise from polyU molecules forming intramolecular hairpin-like structures that consist of hydrogen bonding of U-U base pairs.44, 55, 57 PolyU has minimal secondary structure for an RNA, and this structure is generally present only at low temperature. Our polyU/polyamine cloud point temperature is considerably higher than the temperature at which the polyU structure changed in polyamine-free solution.


12

Page 13 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

Figure 3. Changes in polyU secondary structure are correlated with absorbance in the absence and presence of spermine. In these panels, black traces are normalized absorbance at 260 nm (normalized to the highest absorbance value) and blue traces are turbidity at 500 nm. (A) Normalized polyU absorbance as a function of temperature with no spermine. The absorbance increases up to 12 °C, at which temperature the polyU becomes disordered. (B) Absorbance and turbidity plots for polyU with low spermine (a charge ratio 0.091:1). The transition to disordered polyU rises to 27 °C. The solution does not become turbid at any temperature measured. (C) Absorbance and turbidity for polyU with high spermine (9.1:1 charge ratio) that allows for coacervation. The polyU follows the same trend as the less spermine case, until coacervation begins at 20 °C. The polyU concentration is 0.005% (wt./wt.) in all cases. We next measured the hyperchromicity of polyU in the presence of a low concentration of spermine that allows for formation of soluble complexes of polyU/spermine, but cannot


13

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 14 of 40

induce phase separation (Figure 3B). There was a distinct shift in temperature and magnitude of the change in A260, which indicates that the spermine is capable of stabilizing the structured state of polyU up to ~27 oC, consistent with prior reports for polyU/polyamines.44 In previous studies of polyamine dependent behavior of polyU, the transition temperature was dependent on the identity and concentration of the polyamine,44 as well as buffer and salt identities and concentrations.58 Lastly, we measured the hyperchromicity of polyU with a coacervate-inducing amount of spermine (Figure 3C). Here, we observed the same general shape of the A260 as with polyU and low spermine up to 20 oC. At that temperature, the soluble polyU/spermine complexes began to condense to form the insoluble coacervate droplets (turbidity trace in panel C). This strongly suggests that coacervation with spermine is facilitated by unfolding of the polyU above the cloud point temperature. This could indicate increased accessibility for polyamine binding in the random coil state as compared with the lower-temperature structured form of polyU. Taken together, these results and the prior literature studies of polyU provide some understanding of the molecular structure of polyU and spermine in solution, which is outlined in Scheme 2. At low temperature, polyU has intramolecular secondary structure and base-stacking interactions, and forms soluble complexes with polyamines. When heated above the melting transition temperature (Tm), polyU structure is lost and it adopts a random coil conformation. If present, polyamines stabilize polyU structure and increase Tm. At sufficient polyamine concentrations and temperature above the cloud point (T > Tc), soluble complexes associate together and coacervation occurs. Our system demonstrates that temperature can have a significant impact on coacervation even in systems with very weak secondary structure (polyU), at temperatures well above those to which the structure is unstable in absence of polycation. We note that polyU was chosen for these studies because of its relative lack of secondary structure as


14

Page 15 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

compared to other RNA sequences, and the LCST for RNA/spermine coacervation can be expected to be sequence-dependent. Indeed, prior studies of sequence-dependent binding of spermine to nucleic acids have indicated thermal stabilization on the order of 20-30 oC for a variety of sequences with different degrees of initial secondary structure stability.44

Scheme 2. Temperature-Dependent Structural Changes of PolyU, Stabilized by Spermine, are Important in Coacervation. Compartmentalization of oligonucleotides and peptides within polyU/spermine coacervates. We investigated the compartmentalization of biomolecules within the polyU/spermine coacervates. The propensity of a solute to accumulate within the coacervate is reported in terms of its partitioning coefficient, where K = [concentration in the coacervate phase]/[concentration in the supernatant phase]. Partitioning coefficients were determined for fluorescently-labeled 15nucleotide RNAs as a function of sequence (poly adenylic acid, [poly A15]; poly random base, [poly N15]; and poly uridylic acid, [poly U15]). All of the RNAs accumulated in the droplets


15

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 16 of 40

(K>>1, see Figure 4, Figure S2, and Table 1). The degree of accumulation was sequencedependent, with K = 2800 for poly A15, consistent with its ability to base pair with the long polyU that largely makes up the polyU/spermine coacervate droplets. The poly N15 and poly U15 had K = 62 and 44, respectively, consistent with their reduced ability to interact with polyU and similar to partitioning of single stranded, unstructured RNA in droplets composed of the disordered N-terminus of Ddx4 protein (Ddx4N1).59 In the Ddx4 systems, a 12 nucleotide strand had a K = 33, while a 24 nucleotide strand had K = 39.

59

In a polyU/RRASLRRASL peptide

system, poly A15 also partitioned more strongly than poly N15.15 We note that ion pairing interactions are important in accumulation of charged solutes in complex coacervates, which can occur by substitution for a like-charged coacervate component or by binding an unoccupied oppositely-charged site. The sequence-dependent partitioning observed here shows the importance of additional molecular recognition elements (base pairing in the case of poly A15).

Figure 4. Partitioning of (A) Alexa Fluor 647 poly A15 and (B) TAMRA phosphorylated Kemptide (pKemp) within the polyU/spermine coacervates. Left images are DIC, right are false colored fluorescence. Images of the other oligonucleotides and peptides are in the Supporting Information.


16

Page 17 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

We also measured the partitioning of four different labeled peptides based on the sequence LRRASLG, or Kemptide, which is a model substrate for the enzyme Protein Kinase A60 (Figure 4, Figure S2, and Table 1). It is highly charged with an isoelectric point, or pI, of 12.0 in its dephosphorylated state but has pI = 7.4 in its phosphorylated state (LRRApSLG). Both forms were accumulated within the droplets, with K = 410 and 500 for the dephosphorylated and phosphorylated states, respectively; these values were within error of each other. These peptides partitioned more strongly in the PolyU/spermine than the PolyU/RRASLRRASL system; Kemptide concentration in those droplets was ~240 µM and pKemptide was ~170 µM with the same added concentration in both systems.15 We suspect this is due the peptides’ ability to displace spermine within the droplets better than the larger molecular weight RRASLRRASL peptide. Increasing the peptide length to a 15 amino acid sequence (RRASL)3 and a 25 amino acid sequence (RRASL)5 increased partitioning over the 7 amino acid Kemptide to K = ~730 and ~800, respectively. This can be understood in terms of increased multivalency, allowing release of more counterions upon binding to the polyU. In general, the peptides partitioned much more strongly than proteins in other membraneless organelles, such as the Ddx4N1 organelles. In those droplets, partitioning of 10 different proteins from K = ~0.04 to ~230, with no clear trends based on size or isoelectric point.59 This suggests there is a strong influence of protein structure and/or surface charge distribution61-62 on partitioning in the Ddx4 system, which is not present for peptides in the polyU/spermine system. Using the partitioning data, we calculated the volume of the coacervate phase, which was too small to accurately measure directly. We knew the total volume of the samples (125 µL) and the total number of moles of solute added. With this information and the concentration of the


17

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 18 of 40

solute in each phase, we estimated the volume of the coacervate phase for each solute (Equation S1 and Table S1, Supporting Information). The average volume was 0.09 uL or about 0.07% of the total volume, which was reasonable based on the image of the centrifuged coacervate phase in Figure 1 and the anticipated small volumes of coacervate phase at relatively low total macromolecule contents.51 We calculated the mass added to the solution from the addition of the fluorescent solutes to determine its impact on coacervate volume. In all cases, the added amount of fluorescent solute (0.15 µg or less for the RNAs, 0.42 µg or less for the peptides) was very small compared to 62.5 µg of polyU and 625 µg spermine in 125 µL total volume (Table S1). We concluded that the presence of the fluorescent solutes had a negligible impact on the coacervate volume.


18

Page 19 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

Table 1: Partitioning of Oligonucleotides and Peptides within PolyU/spermine Coacervates* Solute**

Molecular weight (g/mol)

Concent ration added (µM)

Coacervate phase concentration (µM)

Supernatant phase concentration (µM)

Partitioning coefficient (K)

poly A15

5896.3

0.10

70 ± 10

0.025 ± 0.001

2800

poly N15

~5800

0.20

12 ± 2

0.194 ± 0.006

62

poly U15

5550.7

0.20

8.7 ± 0.5

0.20 ± 0.01

44

5550.7

0.20

7.1 ± 0.5

0.197 ± 0.004

36

LRRASLG

1184.35

1.0

410 ± 70

0.95 ± 0.08

432

LRRApSLG

1264.33

1.0

500 ± 100

0.87 ± 0.07

575

(RRASL)3

2181.52

1.0

730 ± 70

0.42 ± 0.03

1738

(RRASL)5

3348.89

1.0

800 ± 200

0.34 ± 0.02

2353

poly

U15

w/

liposomes

* Concentrations were 0.05 % polyU and 0.5% spermine (charge ratio 56:1) ** RNAs were labeled at the 5’ end with Alexa Fluor 647; Peptides were labeled at the N terminus with TAMRA.

Mobility of RNAs within coacervate droplets and exchange with surrounding phase. Fluorescence recovery after photobleaching (FRAP) has become a widely used technique for investigating diffusion inside membraneless organelles.63-65 Here, we used FRAP to compare diffusivity of each of the three RNAs within the coacervate droplets. Two different bleaching


19

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 20 of 40

geometries were used: partial droplet bleaching, in which recovery is largely due to diffusion of molecules within a single coacervate droplet (Figure 5A), as well as entire droplet bleaching, recovery from which necessitates exchange of solute with the dilute continuous phase (Figure 5B). The halftime of recovery (τ1/2) is the time from initial bleaching to the time where the fluorescence has recovered to half of its maximum value. This can be used to compare the relative rates of recovery between the three RNAs. Halftimes of recovery and calculated apparent diffusion coefficients (Dapp) are given in Table 2 for each of the RNAs with both bleaching patterns. For partial droplet bleaching, recovery from within the same coacervate droplet was rapid in all cases, with τ1/2 = 0.5 seconds for poly U15 and 3x slower for poly A15 and poly N15. Poly U15 is expected to have the weakest binding with the much larger polyU, which would allow for a faster recovery compared to the other RNAs. Representative recovery curves for partial droplet bleaching for poly A15, poly N15, and poly U15 are given in Figure 5C-E. Recovery was nearly 100% in all three cases.


20

Page 21 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

Figure 5. Fluorescence images of Alexa Fluor 647-labeled poly U15 RNA demonstrating both partial droplet bleaching (A) and entire droplet bleaching (B) and representative FRAP recovery curves for the fluorescent RNAs (C-H). In (A) and (B), the first images are the droplet before bleaching. The center images are the fluorescence immediately after bleaching, and the right images are the fluorescence at the end of post-bleach recovery. The green circles indicate the bleaching area. Partial droplet recovery for poly A15 (C), poly N15 (D), and poly U15 (E) and full droplet recovery for poly A15 (F), poly N15 (G), and poly U15 (H) are given. Recovery was


21

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 22 of 40

dependent on the surrounding concentration of the RNA as well as binding interactions with the larger polyU. FRAP was done at 37 oC. The concentrations were 0.05 % polyU and 0.5 % spermine (a charge ratio of 56:1). Additional trials are given in Table S2, Supporting Information.

Table 2. FRAP and partitioning data for RNAs within polyU/spermine coacervates Solute

Partitioning coefficient (K)

Poly A15

2800

Poly N15

Poly U15

Poly U15 w/ liposomes

62

44

36

Bleaching region

Halftime of recovery, τ1/2 (s)

Apparent Diffusion Coefficient (µm2s-1)*

Partial

1.6 ± 0.4

0.8 ± 0.3

Entire

290 ± 20

0.005 ± 0.001

Partial

1.6 ± 0.4

0.8 ± 0.2

Entire

23 ± 4

0.05 ± 0.01

Partial

0.5 ± 0.2

4±1

Entire

1.0 ± 0.3

1.9 ± 0.5

Entire w/liposomes

0.5 ± 0.1

2.0 ± 0.4

* Note that because Dapp is calculated based on the fluorescence recovery curve, it is greatly impacted by the availability of unbleached solute; solute compartmentalization can limit recovery and decrease Dapp.65-66

For the cases where the entire droplet was bleached, the halftimes of recovery were more varied. Here, recovery comes from the surrounding supernatant phase, which has a much lower concentration compared to the droplet phase and consequently can limit recovery rates, giving low Dapp compared to partial droplet bleaching. Once again, poly U15 had the fastest recovery. Poly N15 was next; recovery was ~23× slower compared to poly U15. Poly A15 took the longest to recover, at a rate ~13× slower than poly N15 and ~290× slower than poly U15. Representative


22

Page 23 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

recovery curves for bleaching the entire droplet are given in Figure 4F-H. The rate of recovery is dependent not only on the binding interactions as described above for the partial droplet recovery, but also the supernatant phase concentration. Because poly A15 partitions the strongest, the surrounding supernatant phase concentration is the lowest of the three RNAs (25 nM), which decreases the rate of recovery. Poly N15 and poly U15 samples have supernatant phase concentrations that are ~8× more concentrated, and recover more quickly. Recovery was near 100% for poly U15 and approached 90% for poly U15; poly A15 approached 80%. Most reports of FRAP inside membraneless organelles in vivo and in vitro model liquid organelles involve proteins or RNA that are much larger in molecular weight than the short RNA sequences investigated here. However, the recovery times and apparent diffusion coefficients are similar. For example, apparent diffusion of CLN3 mRNA which binds to a droplet forming protein known as Whi3 was CLN3 concentration dependent, in the range of ~0.005 to 0.02 µm2/s.11 Our values are similar; Dapp for poly A15 and poly N15 for entire droplet bleaching was 0.005 µm2/s and 0.05 µm2/s, respectively. Other examples have focused on the proteins within membraneless organelles. For example, an RNA binding protein hnRNPA1 that phase separates to form stress granules had τ1/2 = 2.6 s in reconstituted droplets and τ1/2 = 2.9 s in vivo.8 In another study of 6 different types of droplets each based on disordered regions of proteins within membraneless organelles had halftimes of recovery in the range of 19 to 64 seconds, and recovery fractions ranging from 0.3-0.9.9 Our halftimes of recovery and recovery fractions for the RNAs are similar.


23

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 24 of 40

Interfacial assembly of lipid vesicles Particulate materials can be assembled at liquid-liquid interfaces, even low interfacial tension aqueous-aqueous interfaces, to stabilize droplets and form what are known as Pickering emulsions.67-69 We investigated the interaction of the coacervate droplets with small unilamellar lipid vesicles, ~90 nm in diameter. Figure 6A outlines the experimental approach. Upon addition of the vesicles to a polyU/spermine coacervate system, the vesicles assembled at the coacervate/supernatant phase interface. Figure 6B shows DIC and confocal fluorescence images of the fluorescent vesicles around the polyU/spermine droplets containing fluorescent poly U15. The vesicles were composed of 5 different phospholipids (numbers are mol %, head group functionalization is also indicated): 50% DOPC (choline), 24.2% DOPE (ethanolamine), 24.2% DOPS (serine), 0.1% DOPE-rhod (rhodamine), and 1.5% DOPE-PEG2000 (polyethylene glycol). The ratio of zwitterionic lipids (PC and PE) to negatively charged PS is comparable to what has been reported for the composition of the nuclear membrane: (45-55% PC, 21-27 % PE, 14-21% PS and PI (inositol))70-72. The PEGylated lipid was added at 1.5% (above the reported mushroom-brush transition) to prevent the negatively-charged vesicles from aggregating due to the excess polycation (spermine).73


24

Page 25 of 40

A Vesicles

Coacervate droplets

Vesicle assembly at the interface

B

C

10 µm

Norm. Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

1.0 0.8 0.6 0.4 0.2 0.0

0

4 8 12 Time (s)

5 µm

Figure 6. Vesicle assembly around coacervate droplets. (A) Assembly of lipid vesicles (red circles) around polyU/spermine coacervates (larger dark blue circles). Note that the lipid vesicles are not drawn to scale – vesicle size is exaggerated to emphasize that there are individual vesicles around the droplets and not a continuous membrane. (B) Vesicles (red) assemble at the interface of the coacervate and supernatant phase. Alexa Fluor 647 poly U15 (blue) is also partitioned to the droplets. (C) FRAP recovery curve of entire droplet bleaching of the Alexa Fluor 647 poly U15. We characterized the size and charge of the liposomes using dynamic light scattering and zeta potential measurements of the liposomes, both before and after assembly around the polyU/spermine droplets (Table 3). The liposomes were initially prepared in the 5 mM HEPES buffer with 1 MgCl2. In this buffer, the liposomes were negatively charged, as we expected based on the chosen lipid composition. They were also characterized in supernatant phase, which contains excess spermine, to determine any changes in size and charge. Notably, the liposomes became slightly positively charged, and they increased in size by approximately 10%. All the liposome samples had a low polydispersity index (PDI).


25

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 26 of 40

Table 3. Size and zeta potential of liposomes. Vesicles As prepared in buffer In supernatant phase After membrane formation *Measured at 4 °C

Z-Average diameter at 37 °C (nm)

Polydispersity Index (PDI)

Z-Average diameter at 4 °C (nm)

PDI

Zeta potential at 37 °C (mV)

88 ± 1

0.061 ± 0.009

83.2 ± 0.3

0.056 ± 0.005

-20 ± 6

96 ± 2

0.070 ± 0.009

88.0 ± 0.4

0.12 ± 0.01

+3 ± 3

-

-

86.9 ± 0.4

0.102 ± 0.009

+4 ± 11*

We anticipated that the liposomes would not fuse to form a continuous membrane around the coacervate droplets because the liposomes were PEGylated and (initially) electrostatically repulsive.74 The absence of significant liposome fusion was confirmed by releasing the liposomes after they had been assembled around the droplets for 3 hours. Lowering the temperature of the samples to 4 °C caused dissolution of the coacervates, and allowed us to remeasure the liposomes, which showed no appreciable change in size or zeta potential. We measured the temperature dependence of the coacervate droplets in the presence of liposomes to determine whether their adsorption at the interface stabilized droplets against dissolution. There was no change in the cloud point temperature, indicating no significant stabilization (Figure S3, Supporting Information). FRAP of the lipids showed no appreciable recovery after 15 minutes (Figure S4, Supporting Information), indicating liposomes are immobile at the interface. This is similar to the behavior of liposomes at a neutral polymer/polymer (PEG/dextran) interface.74 Unlike liposomes adsorbed at the interface of a PEG/dextran aqueous biphasic system, where electrostatic repulsions between the vesicles gave rise to strong stabilization against droplet coalescence, here the adsorbed liposomes were essentially neutralized by adsorbed spermine and did not provide effective stabilization against coacervate coalescence. This is to our knowledge


26

Page 27 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

the first time that lipid vesicles have been assembled around complex coacervates. However, amphiphilic oleate molecules have been shown to form multilayers when incubated with polydiallydimethylammonium chloride/ adenosine triphosphate complex coacervates at concentrations below the amphiphile concentration needed to form micelles.18 Finally, we investigated the impact of the assembled liposomes on RNA diffusion into/out of the coacervate droplets. From FRAP analysis of the poly U15, we found that the recovery from entire droplet bleaching was slightly faster compared to the sample without liposomes (Figure 6C). Hence, the presence of the liposomes did not impede exchange of RNA with the continuous phase. The increase in τ1/2 may be explained by a change in partitioning of the poly U15 (K = 36 compared to K=44 without, Table 2). These observations suggest that assembly of intracellular material at the boundaries of membraneless organelles with cytoplasm or nucleoplasm would not necessarily impede rapid exchange of biomolecules between the organelles and their surroundings, nor dissolution of the organelles in response to a phase change.

CONCLUSION In our model system, simple polyamines were capable of condensing the much larger RNA to form liquid droplets that do not require proteins or peptides and that look and behave like the liquid compartments inside cells composed of many different RNA and proteins. RNA is the major component of these compartments, and the temperature-dependent secondary structure of the RNA plays a key role in determining coacervate formation. Specific biorecognition within the liquid compartments (base pairing) determined the degree of partitioning of other non-phase forming RNAs, which remained mobile inside the droplets and could exchange with the


27

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 28 of 40

surrounding solution. Our findings argue for considering not only IDPs but also long, unstructured RNAs as key molecular components that can drive formation of membraneless organelles in vivo. The polyU RNA used here is less complex than the long noncoding RNAs found in vivo. Additional sequence complexity and the presence of RNA-binding proteins could confer additional biorecognition capabilities and tunability of physical properties (e.g., viscosity, thermal and salt response), providing specialization of different types of liquid compartment. Membraneless organelles are characterized by rapid exchange of material with surrounding cytoplasm or nucleoplasm and in many cases by transient nature (e.g., appearing and disappearing during the cell cycle or in response to stress). The focus is generally on these properties, which demonstrate the absence of a continuous membrane separating the interior and exterior phases, rather than a detailed analysis of what material, if any, could exist at the boundary between these organelles and their surroundings. Our diffusivity data show that interfacial accumulation of lipid vesicles did not inhibit exchange of U15 RNA oligonucleotides with the external solution, nor dissolution of the entire droplet in response to cooling. Since particulate materials often assemble at liquid-liquid interfaces, including aqueous-aqueous interfaces, it is reasonable to ask whether intracellular particulates not containing fluorescent labels might accumulate around membraneless organelles in vivo and remain unnoticed. If this occurs in vivo, it would provide an additional organizational motif.

MATERIALS AND METHODS Materials. Polyuridylic acid (polyU) potassium salt (MW 600 – 1,000 kDa, ~2000-3200 bases), spermine tetrahydrochloride, spermidine trihydrochloride, HEPES, HEPES sodium salt, and magnesium chloride hexahydrate were purchased from Sigma-Aldrich (St. Louis, MO).


28

Page 29 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

Nuclease-free water was obtained from Amresco (Solon, OH). HPLC-grade water was purchased from BDH (West Chester, PA). Fluorescent RNA was obtained from Integrated DNA Technologies (Coralville, IA). The oligonucleotides poly A15 (poly(adenylic acid)), poly U15 and RNA random sequence poly N15 were 5’-labeled with Alexa Fluor 647 (NHS ester) through an amino-modifier C6 linkage on the 5’ phosphate. Fluorescently labeled peptides were custom synthesized by GenScript (Piscataway, NJ) as the trifluoroacetic acid salts. The peptide’s amino acid sequences were LRRASLG, LRRApSLG, (RRASL)3, or (RRASL)5. All peptides had a 5carboxytetramethylrhodamine (TAMRA) N-terminus label. Lipids were acquired from Avanti Polar Lipids (Alabaster, Alabama): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2dioleoyl-sn-glycero-3-phosphoethanolamine

(DOPE),

1,2-dioleoyl-sn-glycero-3-phospho-L-

serine (sodium salt) (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (DOPE-rhod), and 1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(polyethylene

glycol)-2000]

(ammonium

salt)

(DOPE-

PEG2000). Lipids were extruded with a Mini Extruder from Avanti Polar Lipids. Polycarbonate extrusion membranes (0.2 µm and 0.05 µm pore size) were purchased from VWR (West Chester, PA). Secure-SealTM, one well spacers (13 mm diameter, 0.12 mm deep) from Life Technologies (Carlsbad, CA) or silicone spacers (9 mm diameter, 2 mm deep) from Electron Microscopy Sciences were used for imaging. 24 × 30 mm micro cover glasses (no. 1.5) were purchased from VWR (West Chester, PA). Slides were silanized as previously described,75 otherwise droplets took on nonspherical geometries during adsorption to the glass slide. All chemicals were used without further purification. Instrumentation. Turbidity measurements were taken on Agilent 8453 diode-array UV-visible spectrometers with Agilent ChemStation Software coupled with Agilent 89090A Peltier


29

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 30 of 40

temperature controllers. Confocal images and FRAP analysis were done using a Leica TCS SP5 laser scanning confocal inverted microscope (LSCM) with Leica LAS AF software and a HCX PL APO CS 63.0×/1.40 oil UV objective (pinhole 1 A.U.). The temperature stage was an Instec TSA021 with an Instec mk1000 or mk2000 temperature controller. Transmitted light images were acquired on a Nikon Eclipse TE-200 inverted microscope with a 100× 0.9 N.A. LU Plan air objective with a PE94 temperature controller and PE100 temperature stage from Linkam Scientific Instruments. Vesicle sizing was determined using dynamic light scattering on a Malvern Zetasizer Nano-ZS; sizing data is reported in terms of the Z-average. Zeta potential measurements of the coacervates and the vesicles were also done on the Malvern Zetasizer Nano-ZS. Coacervate droplet sizing was done using ImageJ. Graphing was done in Igor Pro Version 6.34A software. Coacervate Preparation. A 10 % (wt/wt) stock solution of polyU was prepared in nuclease-free water and was aliquoted for storage at -20°C (pH ~5). The final concentration of polyU for all experiments was 0.05 wt% (~0.5-0.6 µM polyU). Stock solutions of spermine and spermidine were prepared at 10 wt% in HPLC-grade water. Spermine or spermidine was added at a final concentration of 0.5 wt%, corresponding to a final molarity of ~16 mM or ~22 mM, respectively. Fluorescent RNA and fluorescent peptides were dissolved in nuclease-free water. Coacervates were prepared in a 5 mM HEPES, pH 7.6, 1 mM MgCl2 buffer. The pH was measured after the sample was prepared because of the low buffer concentration. The polyU/spermine and polyU/spermidine samples were pH = 7.6. Samples were thoroughly mixed by gentle pipetting. The charge ratio of each sample was found by calculating the number of moles of charges of each polyelectryolyte (1 negative charge from each monomer of polyU, 3.7 positive charges for each spermine molecule, and 2.9 charges for each spermidine molecule).


30

Page 31 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

Characterization of Temperature Dependent Coacervate Formation. The phase transition temperature of polyU/spermine and polyU/spermidine coacervates was determined through UVvisible spectroscopy by measuring the sample absorbance/transmittance at 500 nm and converting to turbidity (100-% Transmittance) or 260 nm and converting to normalized absorbance (A260). The normalization was done using the highest value in the temperature range. The temperature of the sample was ramped from 1°C to 37 °C at various increments, depending on the expected transition temperature. The sample was equilibrated at the desired temperature for 5 minutes before a measurement was taken. At smaller time increments, the cloud point temperature shifted. The data were plotted in Igor Pro Version 6.34A graphing software, which was used to differentiate the data to obtain the dA/dT plots to note the phase transition temperature. Coacervate Imaging. Confocal images were collected with an excitation at 543 nm for rhodamine-labeled lipids or TAMRA-labeled peptides and 633 nm for Alexa Fluor 647-labeled RNA. The temperature was held at 37°C to sustain phase separation. Concentrations were determined through calibration curves of the fluorescently labeled oligonucleotides or peptides prepared in buffer. Coacervate Zeta Potential Measurements. The zeta potential of the coacervates was measured using a disposable folded capillary cell. The measurements were done on undiluted samples immediately following coacervate preparation before extensive coalescence at 0.05% poly U and 0.5% polyamine. Vesicle Preparation and Assembly. Small unilamellar vesicles (SUVs) were prepared by the gentle hydration method39 followed by extrusion through polycarbonate membranes. To mimic the lipid concentration of the nuclear envelope70-72, lipids were added at 2.5 mg/mL at the


31

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 32 of 40

following mole percentages: 50% DOPC, 24.2% DOPE, 24.2% DOPS, 0.1% DOPE-rhodamine, and 1.5% DOPE-PEG2000. The lipids were dried under argon on the surface of a glass test tube, and the sample was placed under vacuum for 1 hour to remove residual solvent. The samples were hydrated in 5 mM HEPES, pH 7.6, 1 mM magnesium chloride buffer and were incubated at 50 °C for at least 12 hours before extrusion following Avanti protocol.76 The lipids were first extruded through 0.2 µm polycarbonate membranes and subsequently 0.05 µm membranes to produce SUVs, passing 11 times through each membrane. The average size and polydispersity of vesicles was determined by dynamic light scattering. Assembly of SUVs around coacervates was achieved by first forming coacervates in a reduced volume (93.75 µL), but with the same mass of polyU and spermine. 31.25 µL of the stock solution of SUVs was added to make a final volume of 125 µL and the same concentration of polyU and spermine as without liposomes. Assuming no lipid loss during extrusion, SUVs final lipid concentration of 0.625 mg/mL. Spectrophotometric and microscopic analyses of coacervates coated with liposomes were conducted as described above. Fluorescent Recovery After Photobleaching. FRAP studies were conducted with excitation at 543 nm for rhodamine-labeled lipids and an excitation of 633 nm for Alexa Fluor 647-labeled RNA (poly A15, poly N15, and poly U15). The temperature was held at 37 °C. After selecting a droplet for analysis, a 10 frame pre-bleach sequence was used followed by a 5 or 10 frame bleach at 100% 458 nm, 476 nm, 488 nm, 514 nm, 543 nm and 633 nm laser power for Alexa Fluor 647-labeled RNA and rhodamine-labeled lipids. Regions of interest (ROI) for partial droplet bleaching and subsequent recovery were 1 µm diameter, from the center of a larger droplet at least 4 µm in diameter. Entire droplet bleaching used the entire diameter of the droplet of interest, ranging from 3-6 µm. For FRAP studies involving assembled liposomes, a square


32

Page 33 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

ROI of 3×3 µm was used to show non-recovery of the lipid fluorescence. For all FRAP studies, a 3 µm diameter circle was used to calculate the noise as a background ROI when all lasers turned off and respective photomultiplier tubes (PMTs) turned on. Recovery was measured by monitoring the fluorescent intensity during the post-bleach sequence. Recovery data was normalized as described by Phair et al.77 and Jia et al.78 through the double normalization method (Equation 1).

FN (t) =

[S(t) − B(t)][R(0) − B(0)] [(R(t) − B(t)][S(0) − B(0)]

(Equation 1)

Here FN ( t ) represents the normalized fluorescence intensity. Three ROIs were monitored at all times 𝑡: S(t) , the average intensity within the arbitrarily chosen sample ROI for bleaching; R(t) , the average intensity of the droplet encompassing the sample bleach ROI (partial droplet diffusivity) or within a similarly sized droplet to serve as a reference ROI that would not be bleached (exchange rate with continuous phase); and B(t) , the average intensity of an arbitrarily chosen background ROI. Position of ROI for each time point was corrected for movements of droplets by using Stackreg plugin79 for A15 entire droplet diffusion. ImageJ software (Rasband, W.S.,

ImageJ,

U.

S.

National

Institutes

of

Health,

Bethesda,

Maryland,

USA,

http://imagej.nih.gov/ij/, 1997-2016) was used for the alignment process and later on to measure fluorescence intensity in ROI. Data was fit to the single exponential recovery function (Equation 2) in Igor Pro Version 6.34A graphing software,

⎛ ⎛ −t ⎞ ⎞ FN (t ) = A ⎜1 − exp ⎜ ⎟ ⎟ + C ⎝ τ ⎠⎠ ⎝

(Equation 2)

where τ is the fluorescence recovery time constant and A and C are constants, with A corresponding to the mobile fraction of fluorescent probe able to recover and C being the y-


33

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 34 of 40

intercept of the recovery curve. The immobile fraction of probe unable to recover corresponds to

1− (A + C) Additionally, the halftime of recovery, τ 1/2 , was calculated from Equation 3.

τ1/2 = τ • ln(2)

(Equation 3)

The apparent diffusion coefficient (Dapp) was calculated by the formula Axelrod et al. described for 2D diffusion,80 where w is radius of bleached area (Equation 4).

Dapp =

0.88w2 4τ 1/2

(Equation 4)

Supplemental Information is available. Absorbance spectra in the UV range of polyU/spermine at different temperatures, additional images of peptide and RNA partitioned in the polyU/spermine coacervates, calculation of the coacervate phase volume and mass of fluorescent solute added, cloud point of polyU/spermine with the vesicles, complete FRAP parameters for each of the RNAs, and FRAP of the assembled lipid vesicles. AUTHOR INFORMATION Corresponding Author *(CDK) email: [email protected] Present Addresses † Present address: Department of Chemistry, Indiana University, Bloomington, IN

47405,

United States ‡ Present address: Department of Chemistry, Waynesburg University, Waynesburg, Pennsylvania Author Contributions


34

Page 35 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

WMA, FPC, and BWD performed the experiments. All authors conceived and designed the experiments and analyzed the data. WMA and CDK wrote the paper, with contributions from FPC and BWD. Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS This work was supported by the National Science Foundation, grant MCB-1244180 (coacervate preparation, temperature dependence, partitioning, liposome interactions) and the NASA Exobiology program, grant NNX13AI01G (FRAP measurements). We thank Philip Bevilacqua for helpful discussions on polyU structure and Erica Frankel for assistance with polyU characterization. REFERENCES 1. Brangwynne, C. P.; Mitchison, T. J.; Hyman, A. A. Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (11), 4334-4339. 2. Brangwynne, C. P.; Eckmann, C. R.; Courson, D. S.; Rybarska, A.; Hoege, C.; Gharakhani, J.; Jülicher, F.; Hyman, A. A. Germline P Granules Are Liquid Droplets That Localize by Controlled Dissolution/Condensation. Science 2009, 324 (5935), 1729-1732. 3. Nott, T. J.; Petsalaki, E.; Farber, P.; Jervis, D.; Fussner, E.; Plochowietz, A.; Craggs, T. D.; Bazett-Jones, D. P.; Pawson, T.; Forman-Kay, J. D.; Baldwin, A. J. Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles. Mol. Cell 2015, 57 (5), 936-947. 4. Elbaum-Garfinkle, S.; Kim, Y.; Szczepaniak, K.; Chen, C. C. H.; Eckmann, C. R.; Myong, S.; Brangwynne, C. P. The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. Proc. Natl. Acad. Sci. U.S.A. 2015, 112 (23), 7189-7194. 5. Berry, J.; Weber, S. C.; Vaidya, N.; Haataja, M.; Brangwynne, C. P. RNA transcription modulates phase transition-driven nuclear body assembly. Proc. Natl. Acad. Sci. U.S.A. 2015, 112 (38), E5237-E5245. 6. Li, P.; Banjade, S.; Cheng, H.-C.; Kim, S.; Chen, B.; Guo, L.; Llaguno, M.; Hollingsworth, J. V.; King, D. S.; Banani, S. F.; Russo, P. S.; Jiang, Q.-X.; Nixon, B. T.; Rosen, M. K. Phase transitions in the assembly of multivalent signalling proteins. Nature 2012, 483 (7389), 336-340.


35

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 36 of 40

7. Wippich, F.; Bodenmiller, B.; Trajkovska, M. G.; Wanka, S.; Aebersold, R.; Pelkmans, L. Dual Specificity Kinase DYRK3 Couples Stress Granule Condensation/Dissolution to mTORC1 Signaling. Cell 2013, 152 (4), 791-805. 8. Molliex, A.; Temirov, J.; Lee, J.; Coughlin, M.; Kanagaraj, A. P.; Kim, H. J.; Mittag, T.; Taylor, J. P. Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization. Cell 2015, 163 (1), 123-133. 9. Lin, Y.; Protter, D. S. W.; Rosen, M. K.; Parker, R. Formation and Maturation of PhaseSeparated Liquid Droplets by RNA-Binding Proteins. Mol. Cell 2015, 60 (2), 208-219. 10. Altmeyer, M.; Neelsen, K. J.; Teloni, F.; Pozdnyakova, I.; Pellegrino, S.; Grofte, M.; Rask, M. B. D.; Streicher, W.; Jungmichel, S.; Nielsen, M. L.; Lukas, J. Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose). Nat. Commun. 2015, 6, 8088. 11. Zhang, H. Y.; Elbaum-Garfinkle, S.; Langdon, E. M.; Taylor, N.; Occhipinti, P.; Bridges, A. A.; Brangwynne, C. P.; Gladfelter, A. S. RNA Controls PolyQ Protein Phase Transitions. Mol. Cell 2015, 60 (2), 220-230. 12. Mitrea, D. M.; Kriwacki, R. W. Phase separation in biology; functional organization of a higher order. Cell Commun. Signal. 2016, 14, 1. 13. Hyman, A. A.; Weber, C. A.; Juelicher, F. Liquid-Liquid Phase Separation in Biology. Annu. Rev. Cell Dev. Biol. 2014, 30, 39-58. 14. Keating, C. D. Aqueous Phase Separation as a Possible Route to Compartmentalization of Biological Molecules. Acc. Chem. Res. 2012, 45 (12), 2114-2124. 15. Aumiller, W. M.; Keating, C. D. Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles. Nat. Chem. 2016, 8 (2), 129-137. 16. Banani, S. F.; Rice, A. M.; Peeples, W. B.; Lin, Y.; Jain, S.; Parker, R.; Rosen, M. K. Compositional Control of Phase-Separated Cellular Bodies. Cell 2016, 10.1016/j.cell.2016.06.010. 17. Aumiller, W. M.; Keating, C. D. Experimental models for dynamic compartmentalization of biomolecules in liquid organelles: Reversible formation and partitioning in aqueous biphasic systems. Adv. Colloid Interface Sci. 2016, doi:10.1016/j.cis.2016.06.011. 18. Tang, T. Y. D.; Hak, C. R. C.; Thompson, A. J.; Kuimova, M. K.; Williams, D. S.; Perriman, A. W.; Mann, S. Fatty acid membrane assembly on coacervate microdroplets as a step towards a hybrid protocell model. Nat. Chem. 2014, 6 (6), 527-533. 19. Koga, S.; Williams, D. S.; Perriman, A. W.; Mann, S. Peptide-nucleotide microdroplets as a step towards a membrane-free protocell model. Nat. Chem. 2011, 3 (9), 720-724. 20. Perry, S. L.; Leon, L.; Hoffmann, K. Q.; Kade, M. J.; Priftis, D.; Black, K. A.; Wong, D.; Klein, R. A.; Pierce, C. F.; Margossian, K. O.; Whitmer, J. K.; Qin, J.; de Pablo, J. J.; Tirrell, M. Chirality-selected phase behaviour in ionic polypeptide complexes. Nat. Commun. 2015, 6, 6052. 21. Perry, S. L.; Li, Y.; Priftis, D.; Leon, L.; Tirrell, M. The Effect of Salt on the Complex Coacervation of Vinyl Polyelectrolytes. Polymers 2014, 6 (6), 1756-1772. 22. Priftis, D.; Tirrell, M. Phase behaviour and complex coacervation of aqueous polypeptide solutions. Soft Matter 2012, 8 (36), 9396-9405. 23. Chollakup, R.; Smitthipong, W.; Eisenbach, C. D.; Tirrell, M. Phase Behavior and Coacervation of Aqueous Poly(acrylic acid)-Poly(allylamine) Solutions. Macromolecules 2010, 43 (5), 2518-2528. 24. Wang, J. T.; Smith, J.; Chen, B. C.; Schmidt, H.; Rasoloson, D.; Paix, A.; Lambrus, B. G.; Calidas, D.; Betzig, E.; Seydoux, G. Regulation of RNA granule dynamics by


36

Page 37 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

phosphorylation of serine-rich, intrinsically-disordered proteins in C-elegans. eLife 2014, 3, :e04591. 25. Brangwynne, C. P.; Tompa, P.; Pappu, R. V. Polymer physics of intracellular phase transitions. Nature Physics 2015, 11 (11), 899-904. 26. Uversky, V. N.; Kuznetsova, I. M.; Turoverov, K. K.; Zaslavsky, B. Intrinsically disordered proteins as crucial constituents of cellular aqueous two phase systems and coacervates. FEBS Lett. 2015, 589 (1), 15-22. 27. Patel, A.; Lee, H. O.; Jawerth, L.; Maharana, S.; Jahnel, M.; Hein, M. Y.; Stoynov, S.; Mahamid, J.; Saha, S.; Franzmann, T. M.; Pozniakovski, A.; Poser, I.; Maghelli, N.; Royer, L. A.; Weigert, M.; Myers, E. W.; Grill, S.; Drechsel, D.; Hyman, A. A.; Alberti, S. A Liquid-toSolid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell 2015, 162 (5), 1066-1077. 28. Quiroz, F. G.; Chilkoti, A. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers. Nat. Mater. 2015, 14 (11), 1164-1172. 29. Mao, Y. T. S.; Zhang, B.; Spector, D. L. Biogenesis and function of nuclear bodies. Trends Genet. 2011, 27 (8), 295-306. 30. Caudron-Herger, M.; Rippe, K. Nuclear architecture by RNA. Curr. Opin. Genet. Dev. 2012, 22 (2), 179-87. 31. Mercer, T. R.; Mattick, J. S. Structure and function of long noncoding RNAs in epigenetic regulation. Nat. Struct. Mol. Biol. 2013, 20 (3), 300-7. 32. Chillon, I.; Marcia, M.; Legiewicz, M.; Liu, F.; Somarowthu, S.; Pyle, A. M. Native Purification and Analysis of Long RNAs. Methods Enzymol. 2015, 558, 3-37. 33. Bond, C. S.; Fox, A. H. Paraspeckles: nuclear bodies built on long noncoding RNA. J. Cell Biol. 2009, 186 (5), 637-644. 34. Bloomfield, V. A. DNA condensation by multivalent cations. Biopolymers 1997, 44 (3), 269-282. 35. Tang, M. X.; Szoka, F. C. The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes. Gene Ther. 1997, 4 (8), 823-832. 36. Arfin, N.; Bohidar, H. B. Condensation, Complex Coacervation, and Overcharging during DNA-Gelatin Interactions in Aqueous Solutions. J. Phys. Chem. B 2012, 116 (44), 1319213199. 37. Leong, K. W.; Mao, H. Q.; Truong-Le, V. L.; Roy, K.; Walsh, S. M.; August, J. T. DNApolycation nanospheres as non-viral gene delivery vehicles. J. Controlled Release 1998, 53 (1-3), 183-193. 38. Mao, H. Q.; Roy, K.; Troung-Le, V. L.; Janes, K. A.; Lin, K. Y.; Wang, Y.; August, J. T.; Leong, K. W. Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. J. Controlled Release 2001, 70 (3), 399-421. 39. Ristori, S.; Ciani, L.; Candiani, G.; Battistini, C.; Frati, A.; Grillo, I.; In, M. Complexing a small interfering RNA with divalent cationic surfactants. Soft Matter 2012, 8 (3), 749-756. 40. Pegg, A. E. The Function of Spermine. IUBMB Life 2014, 66 (1), 8-18. 41. Iarashi, K.; Kashiwagi, K. Modulation of cellular function by polyamines. Int. J. Biochem. Cell Biol. 2010, 42 (1), 39-51. 42. Pelta, J.; Livolant, F.; Sikorav, J. L. DNA aggregation induced by polyamines and cobalthexamine. J. Biol. Chem. 1996, 271 (10), 5656-5662.


37

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 38 of 40

43. Spruijt, E.; Westphal, A. H.; Borst, J. W.; Stuart, M. A. C.; van der Gucht, J. Binodal Compositions of Polyelectrolyte Complexes. Macromolecules 2010, 43 (15), 6476-6484. 44. Szer, W. Effect of Polyamines on Secondary Structure of Synthetic Polyribonucleotides. Ann. N. Y. Acad. Sci. 1970, 171 (3), 801-809. 45. Rowatt, E.; Williams, R. J. The binding of polyamines and magnesium to DNA. J. Inorg. Biochem. 1992, 46 (2), 87-97. 46. Raspaud, E.; Olvera de la Cruz, M.; Sikorav, J. L.; Livolant, F. Precipitation of DNA by polyamines: a polyelectrolyte behavior. Biophys. J. 1998, 74 (1), 381-93. 47. Braunlin, W. H.; Strick, T. J.; Record, M. T., Jr. Equilibrium dialysis studies of polyamine binding to DNA. Biopolymers 1982, 21 (7), 1301-14. 48. Marczynski, B. The binding of spermine to polynucleotides and complementary oligonucleotides at near physiological ionic strength. Int. J. Biochem. 1985, 17 (1), 107-11. 49. Higashi, K.; Terui, Y.; Inomata, E.; Katagiri, D.; Nomura, Y.; Someya, T.; Nishimura, K.; Kashiwagi, K.; Kawai, G.; Igarashi, K. Selective structural change of bulged-out region of double-stranded RNA containing bulged nucleotides by spermidine. Biochem. Biophys. Res. Commun. 2008, 370 (4), 572-7. 50. Jain, S.; Zon, G.; Sundaralingam, M. Base only binding of spermine in the deep groove of the A-DNA octamer d(GTGTACAC). Biochemistry 1989, 28 (6), 2360-4. 51. Frankel, E. A.; Bevilacqua, P. C.; Keating, C. D. Polyamine/Nucleotide Coacervates Provide Strong Compartmentalization of Mg2+, Nucleotides, and RNA. Langmuir 2016, 32 (8), 2041-2049. 52. Vitorazi, L.; Ould-Moussa, N.; Sekar, S.; Fresnais, J.; Loh, W.; Chapel, J. P.; Berret, J. F. Evidence of a two-step process and pathway dependency in the thermodynamics of poly(diallyldimethylammonium chloride)/poly(sodium acrylate) complexation. Soft Matter 2014, 10 (47), 9496-9505. 53. Priftis, D.; Laugel, N.; Tirrell, M. Thermodynamic characterization of polypeptide complex coacervation. Langmuir 2012, 28 (45), 15947-15957. 54. Richards, E. G.; Flessel, C. P.; Fresco, J. R. Polynucleotides. VI. Molecular properties and conformation of polyribouridylic acid. Biopolymers 1963, 1 (5), 431-446. 55. Thrierr, J. C.; Dourlent, M.; Leng, M. A Study of Polyuridylic Acid. J. Mol. Biol. 1971, 58 (3), 815-830. 56. Lipsett, M. N. Evidence for Helical Structure in Polyuridylic Acid. Proc. Natl. Acad. Sci. U.S.A. 1960, 46 (4), 445-446. 57. Young, P. R.; Kallenbach, N. R. Secondary Structure in Polyuridylic Acid Non-classical Hydrogen Bonding and the Function of the Ribose 2'-Hydroxyl Group. J. Mol. Biol. 1978, 126 (3), 467-479. 58. Thrierr, J. C.; Deubel, V.; Leng, M. Interaction between Polyribouridylic Acid and Spermine. Biochimie 1972, 54 (9), 1115-1119. 59. Nott, T. J.; Craggs, T. D.; Baldwin, A. J. Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters. Nat. Chem. 2016, DOI: 10.1038/NCHEM.2519. 60. Kemp, B. E. Phosphorylation of Acyl and Dansyl Derivatives of the Peptide Leu-ArgArg-Ala-Ser-Leu-Gly by the Camp-Dependent Protein-Kinase. J. Biol. Chem. 1980, 255 (7), 2914-2918. 61. Xu, Y.; Mazzawi, M.; Chen, K.; Sun, L.; Dubin, P. L. Protein purification by polyelectrolyte coacervation: influence of protein charge anisotropy on selectivity. Biomacromolecules 2011, 12 (5), 1512-22.


38

Page 39 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Langmuir

62. Obermeyer, A. C.; Mills, C. E.; Dong, X. H.; Flores, R. J.; Olsen, B. D. Complex coacervation of supercharged proteins with polyelectrolytes. Soft Matter 2016, 12 (15), 3570-81. 63. Phair, R. D.; Misteli, T. High mobility of proteins in the mammalian cell nucleus. Nature 2000, 404 (6778), 604-9. 64. Lippincott-Schwartz, J.; Altan-Bonnet, N.; Patterson, G. H. Photobleaching and photoactivation: following protein dynamics in living cells. Nat. Cell Biol. 2003, Suppl, S7-14. 65. Lippincott-Schwartz, J.; Snapp, E.; Kenworthy, A. Studying protein dynamics in living cells. Nat. Rev. Mol. Cell Biol. 2001, 2 (6), 444-56. 66. Sniekers, Y. H.; van Donkelaar, C. C. Determining diffusion coefficients in inhomogeneous tissues using fluorescence recovery after photobleaching. Biophys. J. 2005, 89 (2), 1302-7. 67. Pickering, S. U. Emulsions. J. Chem. Soc. 1907, 91, 2001-2021. 68. Balakrishnan, G.; Nicolai, T.; Benyahia, L.; Durand, D. Particles Trapped at the Droplet Interface in Water-in-Water Emulsions. Langmuir 2012, 28 (14), 5921-5926. 69. Aveyard, R.; Binks, B. P.; Clint, J. H. Emulsions stabilised solely by colloidal particles. Adv. Colloid Interface Sci. 2003, 100, 503-546. 70. Keenan, T. W.; Berezney, R.; Funk, L. K.; Crane, F. L. Lipid Composition of Nuclear Membranes Isolated from Bovine Liver. Biochim. Biophys. Acta 1970, 203 (3), 547-554. 71. Keenan, T. W.; Berezney, R.; Crane, F. L. Lipid Composition of Further Purified Bovine Liver Nuclear Membranes. Lipids 1972, 7 (3), 212-215. 72. Zinser, E.; Sperka-Gottlieb, C. D. M.; Fasch, E. V.; Kohlwein, S. D.; Paltauf, F.; Daum, G. Phospholipid Synthesis and Lipid Composition of Subcellular Membranes in the Unicellular Eukaryote Saccharomyces cerevisiae. J. Bacteriol. 1991, 173 (6), 2026-2034. 73. Marsh, D.; Bartucci, R.; Sportelli, L. Lipid membranes with grafted polymers: physicochemical aspects. Biochim. Biophys. Acta 2003, 1615 (1-2), 33-59. 74. Dewey, D. C.; Strulson, C. A.; Cacace, D. N.; Bevilacqua, P. C.; Keating, C. D. Bioreactor droplets from liposome-stabilized all-aqueous emulsions. Nat. Commun. 2014, 5, 4670. 75. Seed, B. Silanizing glassware. Curr Protoc Immunol 2001, 21, 3K:A.3K.1–A.3K.2. 76. Avanti® Mini-Extruder. http://www.avantilipids.com/index.php?option=com_content&view=article&id=185&Itemid=19 3. (accessed May 20, 2016). 77. Phair, R. D.; Gorski, S. A.; Misteli, T. Measurement of dynamic protein binding to chromatin in vivo, using photobleaching microscopy. Methods Enzymol. 2004, 375, 393-414. 78. Jia, T. Z.; Hentrich, C.; Szostak, J. W. Rapid RNA Exchange in Aqueous Two-Phase System and Coacervate Droplets. Orig. Life Evol. Biosph. 2014, 44 (1), 1-12. 79. Thévenaz, P.; Ruttimann, U. E.; Unser, M. A pyramid approach to subpixel registration based on intensity. IEEE Trans. Image Process. 1998, 7 (1), 27-41. 80. Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W. Mobility Measurement by Analysis of Fluorescence Photobleaching Recovery Kinetics. Biophys. J. 1976, 16 (9), 1055-1069.


39

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 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

Page 40 of 40

RNA/polyamine coacervates

& As s RN tide p pe

Ve s

icle

s

T [NaCl]

No droplets


40

RNA-Based Coacervates as a Model for ... - ACS Publications

Recommend Documents