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Polyamine/Nucleotide Coacervates Provide Strong Compartmentalization of Mg2+, Nucleotides, and RNA Erica A Frankel, Philip C. Bevilacqua, and Christine D. Keating Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04462 • Publication Date (Web): 04 Feb 2016 Downloaded from http://pubs.acs.org on February 8, 2016

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Polyamine/Nucleotide Coacervates Provide Strong Compartmentalization of Mg2+, Nucleotides, and RNA Erica A. Frankel1,2‡, Philip C. Bevilacqua1,2,3*, Christine D. Keating1* 1

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Pennsylvania State University, Department of Chemistry, University Park, PA 16802

Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA 16802

Pennsylvania State University, Department of Biochemistry and Molecular Biology, University Park, PA 16802

KEYWORDS liquid-liquid phase separation, protocell, partitioning, compartmentalization, adenine nucleotides

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ABSTRACT

Phase separation of aqueous solutions containing polyelectrolytes can lead to formation of dense, solute-rich liquid droplets referred to as coacervates, surrounded by a dilute continuous phase of much larger volume. This type of liquid-liquid phase separation is thought to help explain the appearance of polyelectrolyte-rich intracellular droplets in the cytoplasm and nucleoplasm of extant biological cells, and may be relevant to protocellular compartmentalization of nucleic acids on the early Earth. Here we describe complex coacervates formed upon mixing the polycation poly(allylamine) (PAH, 15 kDa) with the anionic nucleotides adenosine 5’ mono-, di, and triphosphate (AMP, ADP, and ATP). Droplet formation was observed over a wide range of pH and MgCl2 concentrations. The nucleotides themselves as well as Mg2+ and RNA oligonucleotides were all extremely concentrated within the coacervates. Nucleotides present at just 2.5 mM in bulk solution had concentrations greater than 1 M inside the coacervate droplets. A solution with a total Mg2+ concentration of 10 mM had 1-5 M Mg2+ in the coacervates, and RNA random sequence (N54) partitioned ~10,000-fold into the coacervates. Coacervate droplets are thus rich in nucleotides, Mg2+, and RNA, providing a medium favorable for generating functional RNAs. Compartmentalization of nucleotides at high concentrations could have facilitated their polymerization to form oligonucleotides, which preferentially accumulate in the droplets. Locally high Mg2+ concentrations could have aided folding and catalysis in an RNA world, making coacervate droplets an appealing platform for exploring protocellular environments.


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1. INTRODUCTION The interior organization of living cells is accomplished by a variety of mechanisms including compartmentalization of ions, molecules and reactions within membrane-bounded or membraneless organelles.1-3 The latter include structures such as the nucleolus, Cajal bodies, and RNA granules, each of which is thought to form by intracellular liquid-liquid phase separation.4-9 Complex coacervation, a type of liquid-liquid phase separation that can spontaneously occur in simple polyelectrolyte-rich aqueous solutions,10 is an attractive means of concentrating macromolecules and has been postulated as a route to both modern-day membraneless organelles and prebiotic compartments on early Earth11-15 Compartmentalization by partitioning into liquid droplets is appealing for early Earth scenarios because of the relative ease of both efficiently encapsulating organic solutes and of entry/egress from the compartment as compared to amphiphile membranes.15-16 Complexation of oppositely-charged polyelectrolytes can result in solutions that contain soluble complexes, solid aggregates, or liquid droplets (complex coacervates), depending on the length and charge density of the polyions and the solution conditions, principally the ionic strength.17-18,15 Since electrostatic interactions are possible for polymers having a wide range of chemical compositions, complex coacervation is relatively robust to molecular identity.10,19-21 Polyelectrolytes with relatively low molecular weights and/or at low concentrations can also form coacervates, suggesting a viable mechanism for compartmentalization on the early Earth, where polymer length and concentration would presumably have been limited.16,22 For example, Mann and coworkers have demonstrated coacervate formation in oligopeptide/nucleotide solutions.16,23-24

Coacervate

droplet

formation

provides

a

simple

mechanism

for

compartmentalization of dilute molecules to generate protocells, drive chemical reactions, and


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aid replication. These non-membranous protocells could have concentrated a variety of relevant molecules such as RNAs, lipids, and peptides, as well as their building block monomers and diverse metal ions.16,25-26 While the molecules that comprise modern intracellular liquid bodies are complex biomacromolecules such as proteins and RNA, for protocell formation less complex and potentially abundant lower-molecular weight polyions are more likely and should be considered. Here, we examine coacervation in aqueous solutions of nucleotides, RNAs, and divalent magnesium ions and partitioning as a function of various solution components. We were interested in testing the range of conditions over which liquid droplets form, and the robustness of these coacervates to various pH and salt conditions, as well as nucleotide identity and concentration. We compare the three adenine nucleotides 5’-AMP, 5’-ADP and 5’-ATP as the anionic species for their ability to direct changes in coacervate formation and partitioning. Previous studies have demonstrated that nucleotides can act as anionic components of complex coacervates, with for example, gum Arabic or polypeptides as their cationic counterparts.12,16,27 Here, we focus on the distribution of nucleotides and RNAs in these systems and the effect and distribution of Mg2+. We held the base constant and varied the phosphate motif (mono-, di-, tri) to change the amount of negative charge on the molecule (see Scheme 1).


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Scheme 1. Structures and pKa values of molecules used to form coacervates. (A) Ionic species for coacervate formation including adenosine nucleotides with one, two, or three terminal phosphate groups and poly(allylamine). (B) Experimental approach for analysis. Reagents are listed in the order added. Nucleotides were added last to prevent coacervates from forming until all of reagents were present. Adenine was chosen as the base owing to its expansive role in modern biology: many enzymes use the dephosphorylation of ATP to ADP as an energy source, and myriad cofactors include adenine such as ATP, S-adenosyl methionine (SAM), flavin adenine dinucleotide (FAD), nicotinamine

adenine

dinucleotide

(NAD),

and

acetyl-coenzyme

A

(acetyl-CoA).28

Poly(allylamine) (or ‘PAH’ for poly(allylamine) hydrochloride) was chosen as the polycation for its chemical simplicity (see Scheme 1) and its known ability to drive complex coacervation with polyanions such as poly(acrylic acid).17,29 PAH is structurally similar to cellular polyamines such as spermidine and spermine, which regulate intracellular RNA processes and with which ATP has been reported to interact.30

31

We chose magnesium chloride as our salt species, as Mg2+ is

required for folding and catalytic activity of many ribozymes.32-35 This divalent metal ion thus provides potential function in the system beyond influencing the ionic strength. We observed that


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formation of these coacervates is robust to changes in pH and Mg2+ concentration, and that partitioning into the coacervate phase results in remarkably high local concentrations of nucleotides, Mg2+, and RNA oligonucleotides.

2. RESULTS 2.1 Coacervate Components and Estimation of Charge Balance Scheme 1 illustrates the experimental approach for coacervate formation. Coacervates of 15 kDa PAH were studied with three nucleotides: adenosine 5´-tri-, di-, and monophosphates (ATP, ADP and AMP), which have different charge density and Mg2+ affinity. Internal phosphates carry a single negative charge, while the terminal phosphate carries either a single or double negative charge, which depends on pH owing to a pKa near neutrality (pKa ~6.8; Scheme 1). ATP has the greatest overall negative charge, between –3 and –4 at neutral pH, while AMP has the lowest, between –1 and –2 at neutrality.36 A PAH concentration of 38.5 μM was generally used, corresponding to a maximum positive charge concentration of 10 mM, i.e. if all ~260 primary amine groups on the PAH are fully protonated (pKa ~9.4). Nucleotide concentrations for a particular experiment were chosen on the basis of charge-matching, assuming maximally charged PAH amine groups and maximally charged phosphates. Although actual charge densities could be lower for both the PAH and nucleotides due to pKa perturbations,37, 38 the assumption of maximal charge was useful in guiding the range of nucleotide and PAH concentrations to be explored for coacervate formation. Accordingly, concentrations for the tri-, di- and monophosphate nucleotides were 2.5, 3.3, and 5 mM, respectively. Samples were compared to each other both under these charge-balanced conditions, as well as nucleotide concentration-


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balanced conditions of 10 mM AMP, 10 mM ADP, or 10 mM ATP that provide excess negative charge for ADP and ATP.

2.2 Characterization of Coacervate Formation Conditions for phase separation for nucleotides with PAH were determined by turbidity measurements as a function of pH and Mg2+ concentration. As shown in Figures 1A and S3, all three nucleotides had an onset of turbidity in the 1-5 mM range when mixed with 38.5 μM PAH, which as mentioned above, corresponds to ~10 mM in amine monomers.

Figure 1. Turbidity of solutions as a function of nucleotide and PAH concentration parametric in nucleotide identity. (A) Turbidity with 38.5 μM PAH (=10 mM in amine moieties) as a function of nucleotide concentration. (B) Turbidity as a function of PAH concentration with 2.5 mM


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ATP (black), 3.3 mM ADP (red) and 5 mM AMP (blue). For all panels, experiments were performed in 5 mM Mg2+ and 2 mM HEPES (pH 7), and error bars are the standard deviation from at least three measurements. Symbols are defined in the legend. Turbidity data as a function of nucleotide and PAH concentration at various pH and Mg2+ can be found in Figures S3 and S4, where they have been fit to an equation for the c½ value, which correlates the concentration of either nucleotide or PAH needed to reach half the maximal turbidity, and the steepness of the transition (equation found in SI). Fits shown above are as per SI Figures and 0 μM PAH points were not included in the fits.

The onset of turbidity, or critical coacervation concentration, was observed at the lowest nucleotide concentration for ATP followed closely by ADP (see log plots in Fig S2), with higher nucleotide concentrations required for AMP. The response of turbidity to nucleotide concentration was steepest for ATP, followed by ADP and then AMP, consistent with their charge densities (see Figure S2). Once sufficient nucleotide concentrations were reached (90% turbidity reached at ~1.4 mM ATP, 1.9 mM ADP and 4.2 mM for AMP; see c½ and n values in Tables S5 and S6), samples remained turbid at nucleotide concentrations up to 25 mM. Turbidity data were nearly identical at pH 5, 7 and 9 (compare Figure S2 panels A-C). We interpret the observed absence of a pH effect as an indication that the deprotonated forms of the ATP, ADP, or AMP phosphate backbones are stabilized by association with Mg2+ and, at low [Mg2+], by association with PAH amine moieties. Tri- and diphosphate nucleotides bind Mg2+ with low dissociation constants around 60 and 670 μM, respectively (Table S1), and hence when Mg2+ is present it can be assumed that nucleotides are bound to Mg2+.35 Concentrations of nucleotide needed to reach a fully turbid sample were similar to those estimated for charge balancing with


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38.5 μM PAH (10 mM in positive charge): maximum turbidity was reached at just under 2.5 mM for ATP, around 3 mM for ADP, and 5 mM for AMP, nucleotide concentration very similar to the estimates above. Plots of turbidity as a function of PAH concentration are provided in Figures 1B and S3 for 5 mM AMP, 3.3 mM ADP, and 2.5 mM ATP, and a variety of Mg2+ concentrations and in Figure S4 for 10 mM each nucleotide at pH 7 and 5 mM Mg2+. Solutions of all three nucleotides were fully turbid between ~10 and 20 μM PAH, and then lost turbidity as PAH concentration increased above 20 μM for AMP and ~50 μM for ADP and ~80 μM for ATP (Figure 1B). These results are consistent with the coacervation of anionic nucleotides and PAH polycations being maximal near charge neutralization, and with the formation of soluble polyelectrolyte complexes under conditions of excess PAH.18 Moreover, this trend has been seen in other systems when PAH is paired with longer polyanions.22,39 The superior ability of ATP and ADP to neutralize PAH as compared with AMP is apparent in the wider range of PAH concentrations that undergo liquid-liquid phase separation for the more highly-charged nucleotides. When higher concentrations of the nucleotides were present (10 mM, Figure S4), samples remained turbid at higher polyamine concentrations, up to at least 100 μM PAH. The appearance of 20% turbidity even before any PAH was added, particularly apparent in 10 mM ADP samples (Figure S4), suggests nucleotide self-association, perhaps driven by nucleotide:Mg2+ complex formation.40 Turbidity could arise from coacervation (formation of liquid droplets) or aggregation (solid particles). To distinguish between these outcomes, samples were imaged by optical microscopy. In most samples that showed turbidity, liquid droplets were present (Figure 2A-E and Supporting Table 2), signifying coacervation. The one exception was AMP samples, which formed solid precipitates at 10 mM AMP (Figure 2F) as well as some 5 mM samples (Figure 2C and


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Supporting Table 2). In summary, coacervate formation is not appreciably affected by pH, as tested from pH 5-9, or by Mg2+, as tested from 0 to 25 mM. This conclusion is supported by the data in Figures 1, 2, S2, and S3 and summarized in Table S2.

Figure 2. Optical microscopy images of PAH/nucleotide coacervate droplets. All solutions were prepared with 38.5 μM PAH, 2 mM HEPES pH 7 and 5 mM Mg2+. A-C. Solutions made with equal charge concentration of 10 mM negative and positive moieties with (A) ATP, (B) ADP, and (C) AMP. D-F. Solutions made from equal nucleotide concentrations of 10 mM with (D) ATP, (E) ADP, (F) AMP. Panels A-E reveal coacervate complexes, while panel F shows an example of an aggregate system. A zoomed image of the droplets is shown in SI Figure 5.

2.3 Coacervate Volumes


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The different sized droplets seen in Figure 2 suggest that different total volumes of the dispersed coacervate phase form under different preparation conditions. We explored this possibility by comparing total coacervate volumes for samples prepared under charge-balanced and concentration-balanced conditions as a function of pH and Mg2+ concentration (Supporting Table 3). Samples were centrifuged at 13,300 g for 10 min to collect the coacervate (or aggregate) phase at the bottom of conical tubes, allowing the volume of coacervate to be estimated by visual comparison to a set of size standards, as detailed in the Supporting Information and illustrated in SI Figure 1. Volume estimates elucidate the phase separation process and facilitate calculation of concentration of solutes in the small volume and high viscosity coacervate (or aggregate) phases. If the relative volumes of the dilute and coacervate phases are known, solute concentrations in the coacervate phase can be determined from solute concentrations in the dilute phase and the total solute added (equation for this determination found in SI). We found that coacervate volumes were small for the ATP and ADP systems, less than 0.3% of the total volume. For instance, solutions with a total volume of 1.00 mL gave rise to an ADPor ATP-rich coacervate phase of 0.5 to 3 µL, depending on pH, NTP and Mg2+ conditions (Supporting Table 3). Notably, even though ADP was present at a higher initial concentration than ATP (3.3 vs 2.5 mM, respectively), ADP had a lower coacervate volume than ATP at every condition analyzed, with ADP coacervate phase volumes ranging from 0.5 to 2 µL and ATP volumes ranging from 1.0 to 3.0 µL. This implies that ADP was more concentrated in the coacervate phase. Several factors influence the volume of coacervate phase formed, including the total amount of phase-forming polyions present and the interaction strength, which depend on electrostatic screening and counterion binding.17


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As mentioned, many of the AMP-containing samples contained aggregates rather than coacervates, particularly those at higher nucleotide concentration (see Figure 2F). The estimated volume of these aggregates ranged from 0.9 to 1.5 % of the total volume Table S3), significantly larger than the volume of the coacervates for ATP or ADP. The large volume of the AMPcontaining aggregates could be due to poor packing of the solid particulates, which might lead to inclusion of some of the dilute phase. We attempted to compress the aggregates by centrifugation for long times and higher g, but no volume compression was apparent. We therefore used the above volume estimates. 2.4 Surface Charge of Coacervate Complexes We performed zeta potential measurements to determine the surface charge for droplets (or aggregates, in the case of 10 mM AMP) in each PAH/nucleotide samples. Surface charge may also provide insight into droplet composition and partitioning properties of the coacervate. Figure 3 displays the surface charge of droplets made from charge-balanced (3A-C) and concentration-balanced solutions (3D-F) as a function of total MgCl2 concentration, parametric in pH.


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Figure 3. Surface charge for coacervate droplets containing 38.5 μM PAH as a function of Mg2+ concentration parametric in pH for all three nucleotides. A-C. Surface charge of (A) 2.5 mM ATP (black), (B) 3.3 mM ADP (red), and (C) 5 mM AMP (blue) containing droplets at pH 5 (2 mM MES), 7 (2 mM HEPES) and 9 (2 mM CHES) as a function of total concentration of MgCl2. D-F. Surface charge of (D) 10 mM ATP, (E) 10 mM ADP, and (F) 10 mM AMP containing droplets at pH 5, 7 and 9 as a function of total concentration of MgCl2. Error bars are the standard deviations for each set of data from three independent measurements. Line traces correspond to pH 5 (circles), 7 (squares), or 9 (triangles).

The surface charge for droplets prepared under charge-balanced conditions (Figure 3A-C) (10 mM PAH monomers and 10 mM total negative charge from 2.5 mM ATP, 3.3 mM ADP or 5 mM AMP) was positive, suggesting excess polyamine at the droplet surface. This is consistent


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with plots in Figure 1A, which indicate that 38.5 µM PAH is greater than needed for maximum turbidity. The ATP-containing coacervates were the most positively-charged, with zeta potentials at ~50 mV; the ADP-containing coacervates were intermediate at ~40 mV, while the AMPcontaining coacervates were lowest at ~20 mV. This trend is likely due in part to the greater availability of ADP and AMP. In all three nucleotide samples, the surface charge is weakly dependent on Mg2+ concentration, with reduced positive charge at higher Mg2+ concentrations. This could be due to increasing ionic strength as more MgCl2 is added to solution, potentially in combination with Mg2+ helping to concentrate nucleotides in the droplets, thereby increasing the net negative charge available at the droplet surface. There is little pH dependence to surface charge, consistent with Mg2+ bound phosphates being fully deprotonated over our entire pH range as discussed above for Figure S2.41 In the concentration-balanced solutions (Figure 3D-F), where nucleotides were at higher concentration of 10 mM each, surface charges ranged from negative to weakly positive (Figure 3D- F). Coacervates made from ATP and ADP showed similar trends to each other (Figures 3DE). They have somewhat negative surface charge (~ –12 mV) at 0 mM Mg2+, suggesting excess nucleotide at the droplet surface, and surface charge increases with Mg2+ concentration, eventually leveling at neutral to slightly positive values around 20 mM Mg2+. The Mg2+ dependence for the concentration-balanced solutions (Fig 3D-F) was opposite that of the chargebalanced solutions (Fig 3A-C), which reflects both the relative availability of nucleotides in these systems and the reduction in absolute value of zeta potential with increasing ionic strength. The AMP-containing samples had quite different dependence on surface charge (compare Figure 3F with D, E). At all Mg2+ concentrations and all pH values tested, the surface charge remains in the range of 0 to ~ +10 mV. AMP has lower charge density and lower Mg2+ affinity as compared to


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ADP and ATP and formed solid aggregates rather than liquid coacervate droplets under these conditions (Figure 2F). Taken together, the zeta potential measurements highlight the tunability of coacervate surface charge with composition. By varying nucleotide identity and concentration and Mg2+ concentration, coacervate surface potentials ranging from + 50 to –12 mV can be achieved.

2.5 Nucleotide Partitioning into the Coacervate Complex coacervates should be enriched in the anionic and cationic components that drive their formation; for example, Spruijt et al report polymer concentrations on the order of 1-3 M for polyacrylic acid/poly(N,N-dimetylaminoethyl methacrylate) coacervates.42 Since PAH has many more charges per molecule than the nucleotides, and nucleotides are present at or above charge-matched concentrations, we expect that nearly all of the PAH present in our samples will be localized within the coacervate phase. In the next three sections, we evaluate partitioning of nucleotides, Mg2+, and RNA oligonucleotide into the coacervate phase. We are particularly interested in evaluating nucleotide concentrations because nucleotide enrichment could have facilitated oligomerization on early Earth.43 Figure 4 provides nucleotide concentrations in the dilute and coacervate phases for charge-balanced conditions, as a function of total Mg2+ concentration, parametric in nucleotide identity. Nucleotide concentrations in the dilute phase were notably lowered upon partitioning, indicating incorporation of substantial concentrations of nucleotide in the small volume coacervates. For example, upon coacervation ATP levels in the dilute phase dropped from ~3-fold from 2.5 mM to ~0.8 mM, while ADP concentration dropped ~5-fold from 3.3 mM to ~0.6 mM at high Mg2+ concentration. The concentration of AMP also dropped somewhat upon coacervate/aggregate formation from 5 mM to ~4 mM; the smaller


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effect is reasonable because it was present at the highest initial concentration and has the lowest charge density. We estimated nucleotide concentrations in the coacervate phase from initial moles minus moles left in the dilute phase upon coacervation, divided by the coacervate volume (Table S2 and Table S4). Concentrations of ATP and ADP in the coacervates were remarkably high (Figure 4 and S6). The concentration of ATP ranged from ~700 mM (no Mg2+) to ~2.5 M (25 mM Mg2+ total) (Figure 4B, black) representing enrichments of ~300- to 1,000-fold, while the concentration of ADP ranged from ~1.2 M (no Mg2+) to ~5 M (25 mM Mg2+ total) representing enrichments of ~350- to ~1,500-fold (Figure 4B, red). We attribute the higher local nucleotide concentration for ADP than ATP to the higher total ADP concentration used, and to the possibility of ADP self-association. Non-zero turbidity values at 10 mM ADP (and, to a lesser extent, also 10 mM AMP) before addition of PAH are consistent with nucleotide self-association (see Figure S4). Formation of ADP dimers or oligomers by self-stacking is facilitated by increasing ADP concentrations and the presence of Mg2+ ions, which reduce intermolecular repulsions.40 Association with PAH may also favor ADP multimerization by this mechanism. ADP self-association could increase molecular packing densities in the coacervates as well as the multivalency of interactions with the polycation. This is not seen in with the ATP because a single ATP can provide more sites of coordination to the Mg2+ and also due to the steric and electrostatic repulsion from the triphosphate species that would occur upon self-association.44


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Figure 4. Nucleotide concentration in (A) dilute and (B) coacervate phases as a function of Mg2+ concentration, parametric in nucleotide identity. Initial concentrations of nucleotides were 2.5 mM ATP (black), 3.3 mM ADP (red) and 5 mM AMP (blue). AMP concentrations in panel B were ~20 mM, independent of the Mg2+ concentration. In addition to nucleotides as noted in the legend, all solutions contained 38.5 μM PAH and were buffered with 2 mM HEPES pH 7. Error bars represent the standard deviation from at least three measurements. Figure S4 displays changes in nucleotide concentration in the coacervate phase with uncertainty in phase volume.

Concentrations of AMP in the coacervates were more modest (Figure 4B, blue). The concentration of AMP in the denser phase (generally aggregates; see Table S2) was ~20-50 mM, representing enrichments of ~4- to ~10-fold and fairly independent of the Mg2+ concentration (Figure 4B); the relatively lower concentrations of AMP in these aggregates and the Mg2+-


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independence suggests weaker interactions with the PAH, consistent with lower charge density, and may also be in part due to inclusions of dilute phase in the solid. Because coacervate and aggregate volumes were estimated by visual inspection, precision was ~ ±0.5 µL for a solution having 1 mL total volume. We therefore also report the concentration estimates based on coacervate volumes either two-fold higher or two-fold lower than the visual inspection value. As shown in Figure S4, changing the coacervate volume by a factor of two did not alter the major finding, i.e. that nucleotides are extremely concentrated within the coacervate phase droplets. We also validated our coacervate nucleotide concentrations by producing a 10x larger volume of 10 mL, where we could more accurately estimate the coacervate volume. We observed a 10 µL phase in a 10 mL sample containing 3.3 mM ADP and 38.5 µM PAH and 5 mM Mg2+, supporting the accuracy of the volumes estimated in the smaller systems.

2.6 Mg2+ ion Partitioning into the Coacervate Coacervates can be enriched in both anionic and cationic species. The preceding section described > 1,000-fold enrichment of ATP and ADP. In this section, we present enrichment of Mg2+ in the coacervate. We determined partitioning of Mg2+ in the PAH/nucleotide coacervates, which is of special interest as high concentrations of Mg2+ are critical for RNA folding and function, especially in the absence of proteins such as on early Earth. The concentration of Mg2+ in both the dilute and coacervate phases was measured as a function of total magnesium chloride added by atomic absorption (AA) spectroscopy (Figures 5 and S7). In both charge-balanced and concentration-balanced conditions, ~50% of the Mg2+ has been depleted from the dilute phase in Figures 5A and 5C. In the coacervate phase, the Mg2+ became very concentrated. In ADP-


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containing coacervates, concentration of Mg2+ reached 4 M in solutions with 3.3 mM ADP and 8.5 M in solutions with 10 mM ADP.

Figure 5. Magnesium ion concentration determined by atomic absorption spectroscopy (AA) in the (A) dilute and (B) coacervate phases of solutions containing equal concentrations of negative and positive charge moieties, and in the (C) dilute and (D) coacervate phases of solutions containing equal concentrations of nucleotides. In addition to nucleotides as noted in the legend, all solutions contained 38.5 μM PAH and were buffered with 2 mM HEPES pH 7. Error bars represent the standard deviation of three or more measurements. Figure S7 displays changes in magnesium concentration in the coacervate phase with uncertainty in phase volume.

In ATP-containing coacervates, the concentration of Mg2+ within the coacervate phase reached a maximum of ~1 M, somewhat lower than in ADP but still very high, while in AMP-containing coacervates, Mg2+ concentrations up to 200 mM were achieved. These extreme Mg2+ concentrations can be attributed to the moderately strong association of the Mg2+ to the available


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nucleotides, along with the very small volume that the coacervates occupy and are consistent with the high local concentrations of the nucleotides found in the coacervate phases described above. We also note that the increase in Mg2+ concentration could also promote the decrease in coacervate volume, as it acts to decrease the electrostatic repulsions between nucleotides in the coacervate phase.

2.7 RNA Partitioning and Concentration into the Coacervate We next evaluated accumulation of RNA oligomers into the PAH/nucleotide coacervate droplets. Such compartmentalization provides a potential mechanism for enriching and allowing the evolution of potentially catalytic RNA or RNA-like molecules, and could have been critical for driving chemical reactions by increased local concentration on the early Earth, especially before highly evolved catalysts. We quantified partitioning of 32P 5’-end labeled N54 by scintillation counting with coacervates made from various pH and nucleotide, and Mg2+ concentrations. Scintillation counting was performed in both charge-balanced and concentration-balanced systems and values are tabulated in SI Table 4. These RNAs represent a mixture of random sequence RNAs of length 54 nucleotides which is thought to be a reasonable length for a ribozyme or aptamer;32 a random sequence was chosen to disfavor sequence bias. The

32

P-

labeled N54 RNA was found to partition strongly into the coacervate in all of the samples analyzed and this finding was robust to a twofold uncertainty in coacervate volume (Figures 6 and S8). For instance, the heterogeneous pool of RNA was found to partition with a – log(K) value of greater than 3 under all conditions of nucleotide identity, pH, and [Mg2+] tested (SI Table 4 and Figure 6). The extreme partitioning of RNA oligonucleotides likely corresponds not only to simple partitioning but also to displacement of nucleotide molecules from the coacervate,


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and can be understood in terms of the greater multivalency for electrostatic interactions between the PAH and these 54-nucleotide RNA sequences as compared with the ATP, ADP and AMP nucleotides.

Figure 6. RNA oligonucleotide partitioning in PAH/nucleotide coacervates measured by scintillation counting. Partitioning of 54-nucleotide RNA into the coacervate phase of solutions prepared with (A) equal charge concentrations and (B) equal nucleotide concentrations. In addition to nucleotides as noted in the legend, all solutions contained 38.5 μM PAH and were buffered with 2 mM HEPES pH 7. Error bars represent the standard deviation from three or more measurements. Under all conditions, the —logK value represents favorable partitioning into the coacervate.

Some differences were observed between charge-balanced and concentration-balanced samples. Under charge-balanced conditions, ADP/PAH and AMP/PAH coacervates had KN54 ~ 10-4 and ATP/PAH had KN54 ~ 10-5; stronger partitioning in the ATP samples could be due in part to the decreased concentration of nucleotide in the sample. In general, partitioning was not dependent on pH or Mg2+ concentration (Table S4). Under concentration-balanced conditions, of 10 mM for all nucleotides, partitioning of the N54 RNA was less strong (Figure 6B). In particular, for ATP/PAH coacervates partitioning decreased from ~10-5 to ~10-2, suggesting that when more


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nucleotides are available, RNA oligonucleotides cannot as readily displace them in the coacervates and supporting the interpretation of the ATP samples above. The change in KN54 for AMP/PAH samples was less than for ATP/PAH samples, consistent with the smaller change in nucleotide concentration (4-fold and 2-fold increase in nucleotide concentration for ATP and AMP, respectively). In contrast to coacervates formed with ATP or AMP, partitioning of N54 RNA in the ADP/PAH coacervates remained largely unchanged (KN54 between ~ 10-3.5 and 10-4.2) upon increasing nucleotide concentration from 3.3 to 10 mM. A notable exception was at very low Mg2+ concentrations and pH 7, where KN54 fell to ~ 10-2. This may reflect the role of Mg2+ in ADP self-association; when Mg2+ is unavailable to aid the multimerization of ADP it behaves more similarly to ATP and AMP. Nonetheless, ADP appears to be more readily displaced by N54 RNA than ATP or AMP when all three are present at the same concentration (Figure 6 and Table S4). These findings underscore the multiple interactions responsible for partitioning in PAH/nucleotide coacervates and point to how coacervate composition dictates local oligonucleotide concentrations, suggesting a potential mechanism by which membraneless organelles could control co-localization and movement of biologically relevant molecules to different intracellular compartments in extant cells. In other aqueous-aqueous biphasic systems such as the polyethyleneglycol(PEG)/dextran system and the coacervates that form in solutions of a disordered tail of the protein, Ddx4,7 partitioning has been shown to depend on nucleic acid sequence length and/or structure. For example, RNA partitioning in PEG/dextran aqueous biphasic system is strongly dependent on length.45 Furthermore, partitioning is affected by differences between double-stranded and single-stranded DNA, with double stranded DNA preferentially partitioning into the PEG-rich phase, while single-stranded DNA partitioned preferentially into the dextran-rich phase.46 Recent


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work from Forman-Kay, Baldwin and coworkers demonstrated accumulation of single-stranded DNA and exclusion of double-stranded DNA by protein-based liquid droplets that mimic Ddx4 membraneless organelles.7 We therefore evaluated the effect of RNA length on partitioning into the PAH/ADP coacervates. PolyA oligonucleotides were prepared in sizes ranging from ~20 nt to ~80 nt by denaturing PAGE. All lengths of polyA showed similar partitioning, with -logK ~5, essentially the same as for the N54 sequences in Figure 6A. We also compared an RNA sequence with no known secondary or tertiary structure (HH16 S) and one with significant amount of secondary structure (HDV E ribozyme) (Figure 7). Differences in secondary structure were confirmed by UV melts (Figure S9). These oligonucleotides also displayed strong partitioning, with no apparent dependence on secondary structure. This independence on RNA sequence, length or structure for partitioning in the PAH/nt system can be rationalized as reflecting the superior ability of all the RNA oligonucleotides to associate with PAH and displace ADP in the coacervates due to their greater multivalency.

Figure 7. PolyA oligonucleotide partitioning compared to partitioning of an unstructured and structured RNA in PAH/ATP coacervates measured by scintillation counting. 32P radiolabeled PolyA RNA was hydrolyzed and fractionated by denaturing PAGE according to size markers.


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Partitioning was analyzed as a function of estimated length (red circles) and compared against the partitioning of an unstructured ribozyme substrate strand (green square) and a structure ribozyme enzyme strand (blue squares). 3. DISCUSSION A key difference between the nucleotide/PAH coacervate system explored here and prior polyamine/nucleotide or polyamine/inorganic phosphate coacervates16,25 is the presence of Mg2+ ions. We included Mg2+ because it may have been prevalent in the early ocean (~0.53 x 10-1 molal).47 It is also known to be important for RNA folding and ribozyme activity. This makes Mg2+ a potentially important factor in “RNA world” scenarios where RNA or RNA-like molecules present on the early Earth provided both catalytic and information storage capabilities.43,48-49 In nucleotide-containing coacervate systems, MgCl2 has two roles: it increases ionic strength that reduces attractive electrostatic interactions between oppositely-charged coacervate-forming molecules, and it binds to nucleotides that reduce the repulsive interactions between them and favor nucleotide self-association. In our experiments, Mg2+ impacted coacervate properties in several ways: (1) increases and decreases in droplet surface charge, (2) decreases in total volume of the coacervate phase, (3) increases in concentration of nucleotides, to as high as 5 M for ADP/PAH, and (4) presence of extremely high concentrations of Mg2+ inside the coacervate droplets up to 8 M. ATP and ADP also became extremely concentrated within the coacervate droplets, ranging from just under 1 M to ~2M (for ATP at 25 mM Mg2+) to higher than 5 M (for ADP at ≥ 10 mM Mg2+). Although uncertainty in our volume estimates could lead to some overestimation of local concentrations, even taking that into account the concentrations for ATP and ADP in the coacervates approaches or exceeds 1 M (for ATP and ADP, respectively, see Figure S6). This is particularly striking for a coacervate given that ATP in


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crystal

structures

with

sodium

ions

and

water

molecules

is

1.2

M,50

and

in

[Mg(HATP)2][Mg(H2O)6] is ~0.9 M.44 We note that local concentrations of nucleotides higher than this are found in some condensed forms of nucleic acids, for example in viral capsids. The bacteriophage lambda virus has a 30 nm diameter capsid that contains a 48300 base pair DNA genome, giving a nucleotide concentration of >11 M51.52 Very high concentrations of solutes in coacervates have some precedents.42 Mann and coworkers reported up to 500 mM ATP in poly(lysine)/ATP coacervates.16 The addition of Mg2+ in our system and the higher charge density of PAH as compared to poly(lysine) both drive nucleotide accumulation within the coacervate phase. The presence of Mg2+ has a particularly large impact on nucleotide accumulation in the coacervates: in the absence of Mg2+, the concentration of ATP in the coacervates is only ~750 mM, similar to that reported for polylysine/ATP,16 while the concentration of ADP in the coacervates is just above 1 M. When Mg2+ is present, ATP and ADP concentrations in their respective coacervates increases with increasing Mg2+, reaching ~2 M and ~ 5M, respectively, for 25 mM Mg2+. We also observe a two- to-fourfold decrease in total coacervate volume as the concentration of Mg2+ is increased to 25 mM, indicating a reduction in the water content. Water content in coacervates is known to vary with the strength of the interactions between polyelectrolytes and is sensitive to salt content.22,53 RNA molecules partitioned strongly into the coacervate droplets, which is as anticipated due to their much greater length as compared to the nucleotides and hence greater multivalency of interaction with the polyamine. In contrast to the nucleotides, partitioning of RNA oligonucleotides into the coacervate phase was relatively insensitive to concentration of Mg2+, remaining high over the entire range tested of 0 – 25 mM. This implies a role for noncovalent


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oligomerization of nucleotides by Mg2+ ions, which is not needed for the N54 RNA strands. Partitioning for the RNA oligonucleotides is consistent with the exchange of ATP, ADP or AMP nucleotides for RNA oligonucleotide strands, owing to the superior ability of oligonucleotides to electrostatically interact with the polyamine. Indeed, nucleic acids can serve as the primary polyanions in coacervate formation.11,16,54 In such systems, longer, more flexible nucleic acids such as single-stranded RNAs or DNAs that lack secondary structure are expected to interact more favorably with polycations than their shorter, more structured, or double-stranded counterparts.55-56 We did not observe differences in partitioning with RNA length or secondary structure in the PAH/ADP coacervate system. This can be understood in light of the relative interaction strength of the polyamines with any of the RNA oligonucleotides as compared with the much smaller ADPs. In the early Earth scenario, such coacervate formation and partitioning preferences could have facilitated synthesis and/or accumulation of increasingly longer RNA oligonucleotides from an external dilute phase. The accumulation of very high local Mg2+ concentrations could have facilitated folding and catalytic activity for RNA or RNA-like molecules on the early earth. Eukaryotic cells have total concentrations of Mg2+ of just 20 mM.57 Although modern catalytic RNAs do not require, nor generally benefit from, molar Mg2+ concentrations, there are intriguing examples of extant ribonucleic proteins (RNPs), like RNase P, in which the catalytic RNA is active in the absence of protein cofactors but only when concentrations of Mg2+ are very high (e.g. 0.3 M Mg2+).58-59 In addition, other large ribozymes including the Group II Intron work best under extreme Mg2+ concentrations (0.1–0.75 M Mg2+).60 Under early Earth scenarios, high concentration Mg2+ may have been useful for folding larger RNA-like molecules to induce catalysis when protein were not available.58-59,61


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4. ASSOCIATED CONTENT Supporting information includes all methods and materials, phase formation and volume summaries, all RNA nucleotide and oligonucleotide partitioning data, and comprehensive figures of turbidity and magnesium concentration in the two phases. This material is available free of charge via the Internet at http://pubs.acs.org AUTHOR INFORMATION Corresponding Author Christine D. Keating; Email: [email protected] Philip C. Bevilacqua; Email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources NASA Exobiology, grant number NNX13AI01G ACKNOWLEDGMENT This work was supported by the NASA Exobiology program, grant number NNX13AI01G. We thank Fatma Pir Cakmak for confocal microscopy assistance. ABBREVIATIONS ATP, Adenosine 5’ triphosphate; ADP, Adenosine 5’ diphosphate; AMP, Adenosine monophosphate; PAH, poly(allylamine).


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