Single stranded RNA motifs for in vitro cotranscriptional production of orthogonal phase separated condensates

Abstract

Protein and RNA-based condensates are emerging as an alternative to classical membrane-bound organelles for the task of compartmentalizing molecules and biochemical reactions. We describe methods for making pure RNA condensates emerging from star-shaped RNA motifs (nanostars) that fold from a single molecule of RNA and interact via kissing loop domains. Through computational design of the nanostar sequences, we demonstrate orthogonal (distinct and immiscible) condensates, which can be individually tracked via fluorogenic aptamers. We also show that aptamers make it possible to recruit peptides and proteins to the condensates with high specificity. We show that nanostars form condensates when assembled from purified and annealed RNA, and when they are being transcribed from synthetic templates at constant physiological temperature. Such orthogonal RNA condensates can be modularly customized and offer a route toward creating systems of functional artificial organelles.

Description

[0001] SINGLE STRANDED RNA MOTIFS FOR IN VITRO COTRANSCRIPTIONAL PRODUCTION OF ORTHOGONAL PHASE SEPARATED CONDENSATES

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. Section 119(e) of copending and commonly-assigned U.S. Provisional Patent Application No. 63 / 588,142, filed October 5, 2023, entitled “SINGLE STRANDED RNA MOTIFS FOR IN VITRO COTRANSCRIPTIONAL PRODUCTION OF ORTHOGONAL PHASE SEPARATED CONDENSATES”, the contents of which is incorporated by reference herein.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0005] This invention was made with government support under 1938194, and 2134772 awarded by the National Science Foundation. The government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] Embodiments of the disclosure concern at least the fields of cell biology and biochemistry.

[0008] BACKGROUND OF THE INVENTION

[0009] Membraneless compartmentalization emerges from the condensation of proteins and RNA, and is recognized as a primary mechanism through which cells dynamically control biochemical processes (1 - 4). By co-localizing nucleic acids, enzymes and metabolites, membraneless organelles (MLOs) such as nucleoli, Cajal bodies and stress granules are believed to regulate biogenesis, transcription, post- transcriptional modification and degradation, and generally improve cellular fitness (4- 7). The emergence of biomolecular condensates has also been linked to disease, particularly to neurodegeneration (8).

[0010] The ability to express “designer condensates” with prescribed properties would be a valuable tool to program cellular behavior (9, 10) and engineer synthetic cells (11). Remarkable examples based on engineered peptides (9, 10) or natural condensate forming RNA sequences (12), have highlighted the feasibility of this concept. The generality of these strategies, however, is hampered by challenges linked to protein engineering and the limited programmability of natural RNA repeats.

[0011] There is a need in the art for methods and materials useful to form synthetic RNA condensates.

[0012] SUMMARY OF THE INVENTION

[0013] Leveraging the algorithmic toolkit of nucleic acid nanotechnology (see, e.g., 13- 15), we have developed systematic methods for expressing designer biomolecular condensates from synthetic RNA nanostructures. In embodiments of the invention disclosed herein, polynucleotides are designed to include elementary motifs that consist of star-shaped junctions, or “nanostars”, which fold co-transcriptionally and assemble thanks to programmable base-pairing interactions, thereby forming liquid or gel-like condensates. Thanks to the selectivity of base pairing, at least three co-existing but fully distinct condensate types can be produced simultaneously. Moreover, expressing the condensates in model synthetic cells generates MLOs with controlled size, number, morphology and composition. In addition, embedding RNA aptamers within the nanostar designs enables selective capture of small molecules and proteins, imitating the ability of natural MLOs to recruit various molecules.

[0014] In embodiments of the invention, single stranded RNA nanostars are transcribed in situ from DNA templates, rather than assembling from pre-synthesized strands. In this context, the described RNA condensate platform is immediately useful for creating a variety of functional MLOs in synthetic cells, and further can be directly applied to engineering living cells. Moreover, embodiments of the RNA condensate platform disclosed herein can exploit the vast design space of RNA nanostars and allows, for example, for the fine tuning of condensate properties and self-assembly behaviors.

[0015] The invention disclosed herein has a number of embodiments. Embodiments of the invention include methods of transcribing a DNA sequence selected to encode a RNA condensate polynucleotide sequence so that the synthetic RNA condensate molecule is formed. In these methods, the sequences of the DNA are designed in a way that allows them to make a synthetic RNA condensate molecule that forms a number of specific structural features, features that result from selected and ordered segments of polynucleotide sequences within the synthetic RNA condensate molecules.

[0016] In illustrative embodiments of the invention, a synthetic RNA condensate molecule comprises 100-300 nucleotides and is formed to include complementary polynucleotide sequences that allow the RNA polynucleotide to self-assemble and form a number of modular molecular architectures that provide unique functionalities. Illustrative features / elements in these synthetic RNA condensate molecules include 3, 4, or 5 arm structures, with each arm comprising a stem and a loop (see, e.g. Figure 1). In these three-dimensional structures, the stem in the arm structures typically comprise at least 10 paired nucleotides and the loops in the arm structures typically comprise at least 9 nucleotides. In addition, in these molecules, the base of the stems in the arm structures typically comprise at least 1, 2 or 3 unpaired linker nucleotides that couple a first arm structure to a second arm structure in these molecules. In addition, a first loop in the synthetic RNA condensate typically comprises at least 4-6 nucleotides selected to be complementary to at least 4-6 nucleotides in a second loop in another synthetic RNA condensate molecule such that the at least 4-6 nucleotides in the first loop of a first synthetic RNA condensate molecule and the at least 4-6 molecules in a second loop of a second synthetic RNA condensate molecule couple together via base pairing so that the synthetic RNA condensate molecules then aggregate, resulting in the formation of RNA condensates (see, e.g. FIG. 1). In certain embodiments of the invention, the synthetic RNA condensate molecule comprises RNA aptamer motifs. Optionally in these methods, the DNA is transcribed within a synthetic cell. Embodiments of the invention include compositions of matter comprising a synthetic RNA condensate molecule disclosed herein (see, e.g., the illustrative working embodiments disclosed in Table S2). A related compositions of the invention comprise An RNA condensate formed by a plurality of aggregated synthetic RNA condensate molecules made by the methods disclosed herein. Additional embodiments of the invention include methods of making a RNA condensate using the synthetic RNA condensate molecules disclosed herein.

[0017] Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1: Condensation of co-transcriptionally folding RNA nanostars, (a) Structure of the RNA motifs, (i) A-type RNA nanostars include a Malachite Green Aptamer (MGA), (ii) B-type include a Broccoli Aptamer (BrA), and (iii) C-type include both aptamers. Variants feature mutually orthogonal, self-complementary (palindromic) Kissing Loops (KLs), whose sequences are shown in the insets. RNA nanostars are transcribed from linear dsDNA templates by T7 RNA polymerase (T7 RNAP). (b) Epifluorescence and confocal micrographs showing condensate formation and coarsening for all three designs in (a) at different timepoints an in vitro transcription reaction. Epifluorescence micrographs have been linearly re-scaled to enhance contrast (see SI Methods in Fabrini et al. Co-transcriptional production of programmable RNA condensates and synthetic organelles. Nat. Nanotechnol. (2024), hereinafter “Fabrini et al ”, section 1.4). Pristine micrographs are shown in Fig. S3 of Fabrini et al. Scale bars are 50 pm. (c) Melting temperatures of A-C condensates, determined as discussed in the SI Methods and Figs S5 and S6 in Fabrini et al. (d) (i) Mean of the chord-length distribution, pCLD, and (ii) average number of condensates per microscopy field-of- view, N, as a function of time. Full chord-length distributions, as extracted from image segmentation, are shown in Fig. S16 of Fabrini et al. (see SI Methods in Fabrini et al., section 1.4). N is not computed for system C, which does not form discrete aggregates. Data are shown as means (solid lines) ± standard deviations (shaded regions) of three field-of-views within one sample.

[0020] Figure 2: Co-transcribed orthogonal RNA nanostars form immiscible condensates of controlled size, (a) Epifluorescence micrographs of binary systems of A and B RNA nanostars (see Fig. 1) at various timepoints during the transcription transient. Different ratios between the concentrations of the two templates, A-T and B- T, are tested, while keeping (A-T)+(B-T) constant, (b) Epifluorescence snapshots analogous to panel (a), but where either A or B is replaced by its non-sticky variant, namely A or B. Sketches on the right hand side show examples of scrambled nonbinding KL sequences, (c) Time evolution of the mean chord-length distribution, pCLD, for samples in (a) and (b), the latter shown in the central panel as dashed lines. See Fig. S16 of Fabrini et al. for full chord-length distributions. Red and cyan curves are relative to A and B condensates, respectively. Data are shown as means (solid / dashed lines) ± standard deviations (shaded regions) of three field-of-views within one sample, (d) Distribution of the radii, rc, of A (red) and B (cyan) condensates as a function of the template concentration ratio (A-T) / (B-T). Epifluorescence micrographs in (a) and (b) have been linearly re-scaled to enhance contrast (see SI Methods in Fabrini et al., section 1.4). Pristine micrographs are shown in Fig. S12 and S14 of Fabrini et al. All scale bars are 50 pm.

[0021] Figure 3: Membrane-less RNA organelles expressed in synthetic cells, (a) Diagram showing membrane-less organelles (MLOs) formed in synthetic cells consisting of water-in-oil emulsion droplets encapsulating transcription machinery and DNA templates, (b) Epifluorescence and confocal micrographs showing MLO formation overtime in synthetic cells expressing (i) A-type, (ii) B-type, and (iii) C-type RNA nanostars (see Fig. 1). Epifluorescence micrographs have been linearly re-scaled to enhance contrast (see SI Methods of Fabrini et al., section 1.4). Pristine images are shown in Fig. S20 of Fabrini et al., alongside images relative to additional time-points, (c) Time-dependent mean of the chord-length distribution, pCLD, computed as discussed in the SI Methods in Fabrini et al. (section 1.4). Data are shown as mean (solid line) ± standard deviation (shaded region) from three fields-of-view within one sample, (d) Scatter plot of condensate volume (VC) vs synthetic cell volume (VSynCell) for samples in (b)-(i) and (b)-(ii). Dashed lines indicate best fits to a linear regression model, with 95% confidence intervals shown as shaded regions (SI Methods of Fabrini et al., section 1.4). MLOs occupy 18.2 ± 0.5% and 15.3 ± 0.3% of the volume of the synthetic cells for A and B systems, respectively, (e) Zoomed-in (top) and larger field-of-view (bottom) confocal micrographs depicting synthetic cells co-expressing A and B-type condensates, with different template concentration ratios (A-T) / (B-T) (compare Fig 2a). (f) Distribution of the ratio between the radii of A and B MLOs (rA / rB) as a function of (A-T) / (B-T) for samples in (e) (see SI Methods of Fabrini et al.), (g) Percentage of synthetic cells containing a given number of A-type (top) or B- type (bottom) MLOs. The percentages of synthetic cells containing only one A and / or only one B MLOs are 78.57%, 97.62%, 86.96%, 77.50%, 87.50% for (A-T) / (B-T) = 0.25, 0.50, 1, 2, 4, respectively. Color codes in panels (f) and (g) match those in (e). Numbers in (f) and (g) indicate sampled synthetic cells, (h) Confocal micrographs showing synthetic cells expressing three orthogonal MLO-forming RNA nanostars (A, B and C in Fig. 1) at different time-points. Scale bars in panel (e), bottom, and panel (h), left and center, are 150 pm. All other scale bars are 50 pm.

[0022] Figure 4: Controlling morphology and composition of membrane-less organelles with linker RNA nanostars, (a) Chimeric RNA linker nanostars (L), with two A and two B KLs, enable control over mixing in systems of A and B nanostars by varying the relative concentration of the linker template L-T. (b) Zoomed-in (top) and larger field-of-view (bottom) confocal micrographs of synthetic cells producing A, B and L nanostars with different ratios between DNA templates ((AT):(L-T):(B-T)). The Linker Fraction, shown in brackets, is computed as (L-T) / ((A-T)+(L-T)+(B-T)). All scale bars are 50 pm. (c) Distributions of mixing indices JA and JB of the MLOs computed as discussed in the SI Methods of Fabrini et al. (section 1.4) and shown as a function of the Linker Fraction for samples in (b). Low JA and JB are indicative of purer A-rich and B-rich phases, while JA, JB ~ 1 indicate complete mixing of the two RNA species. Numbers indicate examined synthetic cells.

[0023] Figure 5: Selective protein capture in designer RNA MLOs. (a) RNA nanostar designs are modified to include a single-stranded 5' overhang that can connect to a protein-binding moiety. Design AYFP is identical to nanostar A (Fig. 1) but can connect to YFP-binding aptamer (YFPapt) (i). Design BSTV is identical to B but can connect to either a streptavidin (STV)-binding aptamer (STVapt) (ii) or a biotinylated DNA oligonucleotide (BiotinDNA) (iii). The templates of protein-binding aptamers (YFPapt-T and STVapt-T) are transcribed in synthetic cells alongside the associated RNA nanostar templates (AYFP-T and BSTV-T, respectively), while pre-synthesized BiotinDNA is encapsulated alongside template BSTV-T. (b) Diagrams (top) and confocal micrographs (bottom) of synthetic cells expressing RNA MLOs in the absence (left) or in the presence of protein binding moieties for the systems presented in (a). When protein-binding aptamers are expressed or the biotinylated DNA oligonucleotide is included, target proteins are recruited in the MLOs. Scale bars are 50 pm (top) and 150 pm (bottom), (c) Protein partitioning parameter, computed from confocal micrographs in (b) as the ratio between the fluorescence signal of the target proteins recorded within and outside the MLOs (see SI Methods of Fabrini et al., section 1.4). Data are shown for the three systems in (a) and (b), with and without protein-binding moieties.

[0024] DETAILED DESCRIPTION OF THE INVENTION

[0025] In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the present invention. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0026] Many of the aspects of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. Certain aspects and embodiments of the invention discussed below (e.g. supplemental (“S”) information) are shown in associated publications, (Fabrini et al. Co-transcriptional production of programmable RNA condensates and synthetic organelles. Nat. Nanotechnol. (2024); https: / / doi.org / 10.1038 / s41565-024-01726-x; hereinafter

[0027] “Fabrini et al.”, and Stewart etal. Modular RNA motifs for orthogonal phase separated compartments. Nat. Commun. (2024) 15.1 : 6244; hereinafter “Stewart et al.”) the contents of each of which is incorporated herein by reference. The following text discusses various embodiments of the invention.

[0028] Condensation of RNA and proteins is central to cellular functions, and the ability to program it would be valuable in synthetic biology and synthetic cell science. Described herein is a modular platform for engineering synthetic RNA condensates from tailor-made, branched RNA nanostructures that fold and assemble co- transcriptionally. Up to three orthogonal condensates can form simultaneously and selectively accumulate guest molecules. The RNA condensates can be expressed within synthetic cells to produce membrane-less organelles with controlled number, size, morphology and compositions, and that display the ability to selectively capture proteins. The in-situ expression of programmable RNA condensates can underpin spatial organization of functionalities in both biological and synthetic cells. As used herein, the term "RNA condensation" refers to RNA molecules that self-assemble or aggregate together to form concentrated, liquid-like compartments within a test tube, cell or the like. These RNA molecules can be modified to interact with proteins and / or small molecules, and recruit them to the condensates.

[0029] Because of the differences in the properties of DNA polynucleotides versus RNA polynucleotides, those of skill in the art cannot use studies for making DNA condensates to predict how to make RNA condensates having specific functional features such as those disclosed herein. Our strategic and empirical studies for doing so are discussed in the sections below.

[0030] As discussed below, embodiments of the invention include methods of making a synthetic RNA condensate molecule. Typically, these methods comprise transcribing a DNA sequence selected to encode a RNA condensate polynucleotide sequence so that the synthetic RNA condensate molecule is formed. In these methods, the sequence of the DNA is selected to transcribe a synthetic RNA condensate molecule that has a number of specific structural features that are encoded by selected and ordered segments of polynucleotides in these molecules (see, e.g. the selected and ordered segments of polynucleotides as illustrated in FIGS. 1, 4 and 5, and in the sequences in Table S2). The structures created by the segments of (at times arbitrarily polynucleotides that are simply complementary and / or palindromic to another segment of polynucleotides) sequences are modular, and can be swapped for one another or other sequences to form a wide variety of polynucleotide molecules that form synthetic RNA condensate molecules.

[0031] In illustrative embodiments of the invention, the synthetic RNA condensate molecule comprises a polynucleotide of 100-300 nucleotides. In addition, the synthetic RNA condensate molecule is formed to comprise complementary polynucleotide sequences that allow the RNA polynucleotide to self-assemble so as to form 3, 4 or 5 arm structures, arm structures comprising a stem and a loop (see, e.g. Figure 1). In these three-dimensional structures, the stem in the arm structures comprise at least 10 paired nucleotides (i.e. are coupled to a complementary stretch of at least 10 paired nucleotides also in the synthetic RNA condensate molecule). The loops in the arm structures typically comprise at least 6 nucleotides. In addition, in these molecules, the base of the stems (distal to the loop) in the arm structures comprise at least 1-3 unpaired linker nucleotides that couple at least 10 paired nucleotides in a first arm structure to at least 10 paired nucleotides in a second arm structure. In addition, in these synthetic RNA condensate molecules, a first loop in the synthetic RNA condensate comprises at least 4-6 nucleotides selected to be complementary to at least 4-6 nucleotides in a second loop in (the same or a different) synthetic RNA condensate molecule such that the at least 4-6 nucleotides in the first loop of a first synthetic RNA condensate molecule and the at least 4-6 molecules in a second loop of a second synthetic RNA condensate molecule couple together via base pairing so that the synthetic RNA condensate molecules aggregate to form a RNA condensate. In some embodiments of the invention, the RNA condensate molecule is further coupled to a protein and / or a nucleic acid. In certain embodiments of the invention, the DNA is a linear dsDNA and / or is transcribed using a purified mRNA polymerase. Optionally in these methods, the DNA is transcribed within a synthetic cell.

[0032] In illustrative working embodiments of the invention, 6 nucleotides in a loop of the molecule comprise a HIV-type Kissing-Loop motif, as shown in Stewart et al. (20), however other sequences are possible, as shown in Fabrini et al. (REF?). In typical methods of the invention, the DNA sequence is created so that the loops are disposed in a single transcribed synthetic RNA condensate molecule, and in addition, the loops in the single synthetic RNA condensate molecule are disposed in a molecular architecture where they should not couple together in a single polynucleotide molecule (e.g. the loops are held by atomic forces distal to each other in the molecular architectural framework of the single polynucleotide molecule).

[0033] In certain embodiments of these methods of making a synthetic RNA condensate molecule, the synthetic RNA condensate molecule comprises a RNA aptamer motif sequence; and / or the synthetic RNA condensate molecule comprises a polynucleotide sequence to which a RNA aptamer couples via base pairing. Optionally, for example, this RNA aptamer motif sequence is disposed in a stem, or can be included as an additional domain on the 3' or 5' end of the condensing RNA polynucleotide; and / or the synthetic RNA condensate molecule comprises a 3' or 5' single stranded overhang (typically of at least 10 or 15 residues), to which a RNA aptamer connects via base pairing. Embodiments of the invention can incorporate a variety of aptamers known in the art such as those described in the RNAapt3D and / or UTexas Aptamer Databases. RNAapt3D is a database of RNA aptamer which contains RNA sequences, their secondary / tertiary structural information, target proteins of RNA aptamers and network of RNA-protein interaction. Ryuma Sato, Koji Suzuki, Yuichi Yasuda, Atsushi Suenaga, Kazuhiko Fukui, "RNAapt3D: RNA aptamer 3D-structural modeling database", Biophys J. 2022 Sep 22;S0006-3495(22)00773-l. The UTexas Aptamer Database includes aptamer sequences, binding affinities, and characteristics of over 1,400 aptamers, as well as a description of the original nucleic acid library, binding buffer conditions, and the DOIs of the original aptamer publications (Askari, A., et al., Nucleic Acids Research, Volume 52, Issue DI, 5 January 2024, Pages D351-D359.

[0034] In certain embodiments of the invention, the methods disclosed herein are designed to form a plurality of synthetic RNA condensate molecules by transcribing a plurality of DNA sequences selected to encode a plurality of RNA condensation sequences (so that a plurality of synthetic RNA condensate molecules are formed).

[0035] Embodiments of the invention include compositions of matter comprising a synthetic RNA condensate molecule disclosed herein (see, e.g. those disclosed in Table S2). For example, one such embodiment of the invention comprises a composition of matter comprising at least one synthetic RNA condensate molecule made by the methods disclosed herein. Optionally these compositions further comprise a protein, for example a protein coupled to the synthetic RNA condensate molecule via a RNA aptamer. In certain compositions of the invention, the synthetic RNA condensate molecule is disposed within a synthetic cell. A related composition of the invention comprises a RNA condensate formed by a plurality of aggregated synthetic RNA condensate molecules made by the methods disclosed herein.

[0036] Additional embodiments of the invention include methods of making a RNA condensate. Typically these methods comprise combining together (e.g. within a synthetic cell) a plurality of synthetic RNA condensate molecules selected to comprise 100-300 nucleotides and further comprise complementary sequences that allow the synthetic RNA condensate molecules to self-assemble so as to form 3, 4, or 5 arm structures comprising a stem and a loop. In such methods, the stem in the arm structures comprise at least 10 paired nucleotides (i.e. are coupled to a complementary stretch of at least 10 paired nucleotides also in the synthetic RNA condensate molecule); the loops in the arm structures comprise at least 6 nucleotides; and the base of the stems (distal to the loop) in the arm structures comprise at least 1-3 unpaired linker nucleotides that couple the at least 10 paired nucleotides in a first arm structure to the at least 10 paired nucleotides in a second arm structure. In such methods, the synthetic RNA condensate molecules are selected to comprise at least a first loop in the arm structures comprises at least 4-6 nucleotides selected to be complementary to at least 4-6 nucleotides in at least a second loop such that the at least 4-6 nucleotides in a first loop of a first synthetic RNA condensate molecule and the at least 4-6 molecules in a second loop of a second synthetic RNA condensate molecule couple together so that the synthetic RNA condensate molecules aggregate to form a RNA condensate. In these methods, the plurality of synthetic RNA condensate molecules are combined under aqueous conditions selected to facilitate condensation (e.g. physiological conditions). These methods further include allowing the plurality of synthetic RNA condensate molecules to aggregate so that the RNA condensate is made. In some embodiments of these methods, the plurality of synthetic RNA condensate molecules comprise a homogeneous population of polynucleotides. In other embodiments of these methods, the plurality of synthetic RNA condensate molecules comprise a heterogeneous population of polynucleotides. In certain embodiments of these the plurality of synthetic RNA condensate molecules are further coupled to proteins and / or nucleic acids.

[0037] Illustrative working embodiments of the invention are shown in the figures and discussed below. The four-armed RNA nanostars, shown in Fig. la, consist of a single RNA strand that folds co-transcriptionally into the intended shape. The star-shape was inspired by well characterized DNA nanostars (17-19). However, the RNA motifs interact via self-complementary Kissing-Loops (KLs) present at the end of each arm (20, REF to Fabrini et al.), rather than via the single-stranded (ss) overhangs or hydrophobic modifications adopted for DNA designs (17-19). We tested a dozens of RNA nanostar designs in Stewart et al. and Fabrini et al. Stewart et al. characterizes condensate formation starting using (palindromic) HIV-type KLs that include mutations, as well as non-palindromic KLs that can be introduced in multi -nanostar systems. Here we illustrate the development of a system of three RNA nanostars labelled A, B and C, featuring mutually orthogonal KL sequences (see zoomed-in boxes in Fig. la). In designs A and B, one of the dsRNA arms includes a Fluorescent Light up Aptamer (FLAP): Malachite Green Aptamer (MGA) for A (21, 22) and Broccoli Aptamer (BrA) for B (23) (Figs la (i) and (ii)). FLAPs yield a fluorescent signal upon binding their cognate fluorophores (malachite green for MGA and DFHBI for BrA), enabling characterization via fluorescence microscopy and fluorimetry. In design C, both MGA and BrA were included in non-adjacent arms (Fig. la (iii)). The fluorescence characteristics of all RNA constructs are reported in Fig. SI in Fabrini et al. The arms that do not host FLAPs were designed to be 25 base-pairs long, and separated by an unbound uracil residue at the junction to improve flexibility (24). The motifs were designed to be transcribed with T7 RNA Polymerase (T7 RNAP) from fully doublestranded DNA (dsDNA) templates, labelled as A-T, B-T and C-T for designs A, B and C, respectively (see Fig. S2 in Fabrini et al.). Details on sequence design and in vitro transcription protocols are reported in SI Methods in Fabrini et al., sections 1.1-1.3. All sequences are reported in Tables SI -S3 in Fabrini et al.

[0038] As shown in Fig. lb with representative epifluorescence and confocal snapshots, all three designs formed aggregates during in vitro transcription experiments. Additional examples are provided in Fig. S3, Fig. S4 (top) and video SI (top) in Fabrini et al. Variants A and B formed spherical condensates that nucleated and grew, with frequent coalescence events, confirming that these designs yield liquid-like condensates. Conversely, design C formed a gel-like percolating structure which failed to produce discrete condensates but still grew over time. The higher apparent viscosity of design C when compared to A and B correlates with the thermal stability of the materials. Indeed, C exhibited the highest melting temperature, followed by B and A (Figs 1c, S5 and S6, and video S2 in Fabrini et al.). Regardless of their morphology, all aggregates displayed the intended fluorescence output, namely in the MGA channel for A (red), BrA channel for B (cyan) and both channels for C (white).

[0039] Degradation of the condensates resulting in bubble formation was observed over time, particularly for A-type aggregates (Fig. S3 in Fabrini et al.). This is ascribed to the action of environmental nucleases (25) of photodegradation.

[0040] The specificity of KL interactions was confirmed by non-sticky, control designs where KLs were replaced with scrambled sequences ( A, B). These designs did not yield condensates and only produced diffused fluorescence, as shown in Figs S4 (bottom) and S7, and video SI (bottom) in Fabrini et al.

[0041] Besides microscopy visualization, aptamer fluorescence was also used to monitor the rate of synthesis of RNA building blocks with fluorimetry, as reported in Fig. S8 in Fabrini et al.

[0042] All designs displayed a rapid initial increase, whose rate scaled (nearly) linearly with template concentration (Figs S9 and S10 in Fabrini et al.). After the initial phase of rapid RNA production, synthesis slowed down significantly or plateaued depending on the specific design, due to loss of polymerase activity and / or nucleotide depletion (26-28). A peak was noted for condensate-forming motifs (A, B and C), which was not present in non-sticky designs ( A and B), ascribed to sedimentation of the fluorescent aggregates below the measurement plane. Differences in plateauing behaviors could be due to variations in the kinetics of aptamer folding and / or complexation with the respective fluorophores (29-31).

[0043] We quantified the aggregate growth dynamics for all designs by measuring the temporal evolution of the mean chord length pCLD, as extracted from segmentation of epifluorescence timelapses (see SI Methods in Fabrini et al., section 1.4). pCLD provides a measure of the typical length scale of the fluorescent features, and was chosen because it is applicable and meaningful regardless of aggregate shape (32-34). As shown in Fig. Id (i), aggregate size increased rapidly at early stages for all designs, before plateauing for A and B. Condensate size plateauing occurred roughly when fluorimetry traces also saturated (Fig. S8 in Fabrini et al.). The correspondence of these timescales hints at a scenario in which condensate growth slows down or stops when monomer transcription ceases, and coalescence of large condensates becomes rare, owing to the lack of substantial Brownian motion (video SI in Fabrini et al.). For design C, the increase in typical aggregate length scale continued at later times, driven by coarsening of the percolating RNA network. Full chord-length distributions (CLDs) are shown in Fig. SI 1 in Fabrini et al.

[0044] Trends consistent to those noted for pCLD were found for the average number, N, of A- or B-type condensates counted in each microscopy field-of-view. Data summarized in Fig. Id (ii) show that N monotonically decreased following condensate coalescence. The number of aggregates was not studied for design C given its tendency to form a single percolating structure.

[0045] KL orthogonality enables the simultaneous transcription and assembly of designs A and B, that readily formed distinct, co-existing condensates, as shown in Fig. 2a (see also Figs S12, S13, and video S3 in Fabrini et al.). Consistently, if one of the RNA motifs was rendered non-sticky, condensates of one species co-existed with dispersed RNA nanostars of the other, as shown in Figs 2b, S14, SI 5, and video S4 in Fabrini et al.

[0046] A simple strategy to control the relative size of A and B condensates is to tune the ratio between the concentrations of the corresponding DNA templates ((A-T) and (B-T), respectively), which determines the production rate of each nanostar, as demonstrated in Figs S9 and S10 in Fabrini et al. Relative-size control is demonstrated visually in Fig. 2a (see also Figs S12 and S13 in Fabrini et al.), through time-dependent pCLD analysis in Fig. 2c (see Fig. S16 in Fabrini et al. for full CLDs), and through the distribution of the final radii rc of the condensates, shown in Fig. 2d (see SI Methods in Fabrini et al., section 1.4). We observed condensate number to anti-correlate with their size. For instance, many small A-type condensates were formed when (B-T)>(A- T) (see Fig. S17 in Fabrini et al.).

[0047] As shown in Fig. 2c, condensates were found to grow in two stages in all binary systems of sticky nanostars: after an initial increase, a brief intermediate plateau was reached, followed by another growth phase before saturation. This behavior was not observed in single component systems (Fig. Id), nor in binary systems where one nanostar population was rendered non-sticky, for which the temporal evolution of pCLD is included in the central panel of Fig. 2c. Inspection of the relevant microscopy videos (videos S3 and S4 in Fabrini et al.) reveals that the intermediate size plateau was reached when condensates became temporarily hindered in their ability to make contact with same-type assemblies, due to being “caged” by nearest-neighbors condensates of the opposite type. Coalescence events that still managed to occur, however, reduced lateral crowding given that merged condensates occupy less space in the horizontal plane, triggering a positive-feedback process that rapidly unjammed the system and accelerated coalescence. In fact, Fig. 2c shows that condensates reached a larger overall size in systems where both A and B were sticky, compared to A & B and A & B mixtures (Figs 2b, S14 and S15 in Fabrini et al.), indicating that steric encumbrance from non-binding condensates of the opposite phase ultimately facilitates coalescence.

[0048] Two-step decreasing trends, consistent to those seen in pCLD, were observed in the number of condensates per field-of-view (Fig. S17 in Fabrini et al.).

[0049] Condensate co-assembly is also possible with all three RNA species A, B, and C, as shown in Figs S 18, S19 and video S5 in Fabrini et al. After allowing sufficient time for relaxation, all three species formed distinct spherical condensates, including C, which was unable to do so in single-component samples (Fig. lb (iii)). The difference in morphology is likely due to A and B stars hindering the formation of a percolating C network in favor of smaller aggregates that relaxed more readily.

[0050] Addressable self-assembled RNA condensates could be extremely valuable to engineer compartmentalization in synthetic or living cells, where they could operate as membrane-less organelles capable of recruiting target compounds and underpinning spatial separation of functionalities. To demonstrate this, we transcribed our condensates within basic synthetic cells constructed from water-in-oil emulsion droplets (Fig. 3 a). All designs formed condensates that equilibrated into a single spherical organelle in each synthetic cell (Figs 3b and S20 in Fabrini et al.), including design C that generated only extended networks when produced in bulk. The different morphology is readily rationalized by noting that, when confined to the synthetic cell, C aggregates only need to relax over much smaller length scales. Yet, shape relaxation was slower for C compared to A and B, as confirmed by the time evolution of pCLD in Fig. 3c (see also micrographs in Fig. S21 (top) and full CLDs in Fig. S22 in Fabrini et al.) and video S6 (top) in Fabrini et al.). Larger variation in condensate size reflects the variability in the size of droplet-based synthetic cells produced via emulsion methods. We indeed observed that the final volume of the RNA condensates scales linearly with the volume of the surrounding emulsion droplet, as presented in Fig. 3d.

[0051] Control experiments with non-sticky designs revealed uniform fluorescence within the synthetic cells, confirming that the assembly specificity previously noted in bulk samples persists in confinement (Figs S21 (bottom), S23, and video S6 (bottom) in Fabrini et al.). Like in bulk experiments, fluorimetry can be used to monitor RNA synthesis rates, as shown in Fig. S24 in Fabrini et al., where the delayed growth in the MGA signal (A component) is due to initial accumulation of the malachite green fhiorophore in the oil phase (35, 36), rather than to a slower growth of the condensates (compare with Fig. 3c). Also consistent with trends recorded in the bulk, initial transcription rates as determined via fluorimetry were found to scale (nearly) linearly with template concentration (Figs S25 and S26 in Fabrini et al.).

[0052] We obtained synthetic cells with two distinct synthetic organelles by encapsulating both A-T and B-T templates, as shown in Figs 3e, S27-S31, and videos S7-S8 with microscopy, and Fig. S32 with fluorimetry in Fabrini et al. Like in the bulk experiments, we could control the relative size of the organelles by changing the template ratio, as quantified in Fig. 3f for synthetic cells containing exactly one A and one B condensate. In the vast majority of cases, in fact, each synthetic cell contained one condensate of each type (Fig. 3g). When including component C, we obtained 3 distinct phases, as demonstrated by microscopy in Fig. 3h, (see also Fig. S33, video S9 in Fabrini et al.) and fluorimetry (Fig. S35 in Fabrini et al.), exemplifying the possibility for scaling up the number of addressable organelles. Consistent with bulk experiments (Figs S18 and S19 in Fabrini et al.), relaxation timescales were significantly slower for the ternary system compared to binary ones (Figs 3e and S33 in Fabrini et al.). It is also evident that the synthetic cells often failed to produce exactly three distinct MLOs, likely due to steric effects and the intrinsic slow relaxation of phase C. When replacing component C with ~C, which features identical KLs but lacks any FLAPs, synthetic cells with exactly three MLOs were more common (Fig. S34 in Fabrini et al.), suggesting that the presence of the aptamers has an effect on the coarsening kinetics of the material.

[0053] We further expanded the possible organelle architectures in the synthetic cells by introducing linker RNA nanostars, dubbed L, that could control the mixing between the A and B designs upon their simultaneous transcription. Nanostar L is “chimeric”, as it features two A-type (37) and two B-type KLs (Fig. 4a). As shown in Fig. 4b, low fractions of the linker template ((A-T):(L-T):(B-T)=10:l : 10, or Linker Fraction = 1 / 21) produced grape-like clusters by inducing adhesion between condensates rich in A and B, while blocking their relaxation into two large condensates. Arrested coarsening is arguably due to inter-phase adhesion limiting the ability of the condensates to slide past each other. At higher Linker Fractions, the two opposite-type droplets coarsened into bigger domains, with Janus, capsule-like, and Russian-doll morphologies emerging at (A-T):(L-T):(B-T) = 5: 1 :5 (Linker Fraction = 1 / 11), 3: 1 :3 (Linker Fraction = 1 / 7) and 2: 1 :2 (Linker Fraction = 1 / 5), respectively (see also Figs S36-S40 and videos S10, Si l in Fabrini et al.).

[0054] For the latter, the fact that the A-phase formed the outer shell is consistent with the observation of a lower melting point compared to B, indicating weaker KL interactions and arguably lower interfacial tension with the bulk solution. For linker fractions of 1 / 3 and above, single phase spherical condensates were observed. The abundance of linker nanostars also influences the degree of mixing of the two phases, assessed by measuring indices JA and JB, computed as the ratios between intensity of the fluorescent signal from the minority RNA component (e.g. signal from component A in B-rich condensates) over that recorded for the intensity of the majority phase (e.g. signal from A in A-rich condensates), as outlined in the SI Methods in Fabrini et al. ( section 1.3). The data, collated in Fig. 4i show a little mixing (JA, JB « 1) for low linker fraction, followed by moderate increase in mixing when more linker was produced and by an abrupt jump to JA, JB ~ 1 upon reaching the threshold for complete mixing.

[0055] While the data in Figs 1-4 demonstrate that RNA condensates can selectively sequester small molecules, namely the fluorophores associated to MGA and BrA, imitating the functions of natural MLOs requires capturing larger and more functionally relevant macromolecules, particularly proteins. To this end, we modified designs A and B to include a 5' overhang, to which a protein-binding RNA aptamer can connect via base pairing (Fig. 5a (i) and (ii)). In particular, the new nanostructures AYFP and BSTV were designed to connect to a YFP -binding aptamer (YFPapt) (38), and a streptavidin (STV)-binding aptamer (STVapt) (39), respectively (see also Fig. S41 in Fabrini et al.). For both designs, when co-expressing the modified nanostars and their partner aptamers from distinct templates in synthetic cells (Fig. S42 in Fabrini et al.), the target proteins (EYFP and Alexa405-STV) readily partitioned within the formed MLOs (Figs 5b (i) and (ii), S43, S44a, and video S12 inFabrini et al.). If the aptamer template was omitted, the target proteins remained in the lumen of the droplet-based synthetic cell. Protein partitioning was quantified through the parameter computed from image segmentation as the ratio between the fluorescence intensity of the protein recorded within or outside the MLOs (SI Methods in Fabrini et al. section 1.3). When proteinbinding aptamers were present, S, exceeded 3 and 2 for YFP and STV, respectively, while it fell below 0.5 when no aptamers were present (Fig. 5c (i) and (ii)).

[0056] As an alternative strategy for STV capture, we replaced STVapt with a biotinylated DNA oligonucleotide complementary to the overhang in nanostar BSTV (Figs 5a (iii), S43, S44 and video S12 in Fabrini et al.). This approach still enables B- type MLOs to capture STV (Figs 5b (iii) and c (iii)), with the difference that the protein was distributed non-uniformly within the RNA condensates. Indeed, we observed irregularly-shaped clusters with solid-like appearance, reminiscent of multi-phase condensates found in cells (40-42). The non-uniform protein distribution likely results from the finite amount of the biotinylated DNA anchor available, which is all sequestered at early transcription stages and thus accumulates at the center of the condensates. The solid-like look of the protein-rich material may be a consequence of the tetravalent STV, which can irreversibly crosslink up to four RNA nanostars and thus make the material more viscous.

[0057] The platform disclosed herein enables the expression of synthetic condensates and membrane-less organelles with prescribed size, number, morphology, composition, and the ability to capture small guest molecules and proteins. The elementary building blocks, RNA nanostars, were designed from first principles utilizing the rule-based approaches of nucleic acid nanotechnology, which provide extensive opportunities for further design variations aimed at programming arbitrary characteristics of the designer MLOs. Among the many design features that can be straightforwardly controlled are nanostar valency, flexibility and arm-length, all known to predictably influence selfassembly in analogous DNA systems (17, 19, 24, 29, 43). Further RNA aptamers might be embedded to recruit molecular guests, including enzymes and metabolites, while ribozymes (44) could confer catalytic properties to the synthetic MLOs. Because the condensates can be expressed from DNA templates, their formation could be controlled through standard transcription regulation pathways in both synthetic cells and living cells. Owing to their open-ended programmability, we expect that the RNA-nanostar condensates will constitute a valuable new solution for the toolkit of synthetic biology.

[0058] TABLE SI: SEQUENCES OF ILLUSTRATIVE RNA NANOSTARS, REPORTED ACCORDING TO 5’-3’ CONVENTION. 1. A’

[0059] GCA CAG UGC UAU GAG UGU GCA CGG GAU CCC GAC UGG CGC CAA CGA ACA AGG UGC CAG GUA ACG AAU GGA UCC UGU GCU GCA CAU UAG AGU CGC UGU AUG ACC CAU AGC ACA AGG GUC GUA

[0060] CAG CGG CUC UAG UGU GCU CGC GUG CCU CAG AGG ACC UGU CAC

[0061] CAU GGC GAA AGG UGA UAG GUC CUU UGA GGU ACG CGU CAC

[0062] UCG UAG CAU UGU GCC UGU CUC CAA CGG CAA AGG AGA UAG (SEQ ID NO: 1)

[0063] 2. A

[0064] GCA CAG UGC UAU GAG UGU GCA CGG GAU CCC GAC UGG CCG CAU CGC GAA AGU GGC CAG GUA ACG AAU GGA UCC UGU GCU GCA CAU UAG AGU CGC UGU AUG ACC CAU CGC GAA AGG GUC GUA CAG CGG CUC UAG UGU GCU CGC GUG CCU CAG AGG ACC UGU CAC CAU CGC GAA AGG UGA UAG GUC CUU UGA GGU ACG CGU CAC UCG UAG CAU UGU GCC UGU CUC CAU CGC GAA AGG

[0065] AGA UAG (SEQ ID NO: 2)

[0066] 3. B’

[0067] GCA CAG UGC UAU GAG UGU CGC GAC GGA GAC GGU CGG GUC CAG AUA GGC CAU GGC GAA AGG UCU AUC UGU CGA GUA GAG UGU GGG CUC CGU CGC GUG CAC AUU AGA GUC GCU GUA UGA CCC AUA GCA CAA GGG UCG UAC AGC GGC UCU AGU GUG CUG CAC AGU GUC UGU GCG ACU GCA CCC AAC GAA CAA GGG UGU AGU CGC AUA GAC AUU GUG CUC ACU CGU AGC AUU GUG CCU

[0068] GUC UCC AAC GGC AAA GGA GAU AG (SEQ ID NO: 3) 4. B

[0069] GCA CAG UGC UAU GAG UGU CGC GAC GGA GAC GGU CGG GUC CAG AUA GGC CAG UCG ACA AGG UCU AUC UGU CGA GUA GAG UGU GGG CUC CGU CGC GUG CAC AUU AGA GUC GCU GUA UGC C AC AGU CGA CAA GUG GCG UAC AGC GGC UCU AGU GUG CUG CAC AGU GUC UGU GCG ACU GCA CCC AGU CGA CAA GGG UGU AGU CGC AUA GAC AUU GUG CUC ACU CGU AGC AUU GUG CCU GUC UCC AGU CGA CAA GGA GAU AG (SEQ ID NO: 4)

[0070] 5. C

[0071] GCA CAG UGC UAU GAG UGU CGC GAC GGA GAC GGU CGG GUC CAG AUA GGC CAG GUA CCA AGG UCU AUC UGU CGA GUA GAG UGU GGG CUC CGU CGC GUG CAC AUU AGA GUC GCU GUA UGC CAC AGG UAC CAA GUG GCG UAC AGC GGC UCU AGU GUG CUG CAC GGG AUC CCG ACU GGC CGC AGG UAC CAA GUG GCC AGG UAA CGA AUG GAU CCU GUG CUC ACU CGU AGC AUU GUG CCU GUC UCC AGG UAC CAA GGA GAU AG (SEQ ID NO: 5)

[0072] 6. C’

[0073] GCA CAG UGC UAU GAG UGU GCA CAG UGU CUG UGC GAC UGC ACG CAG GUA CCA AGC GUG UAG UCG CAU AGA CAU UGU GCU GCA CAU UAG AGU CGC UGU AUG ACC CAG GUA CCA AGG GUC GUA CAG CGG CUC UAG UGU GCU CGC GUG CCU CAG AGG ACC UGU CAG CAG GUA CCA AGC UGA UAG GUC CUU UGA GGU ACG CGU CAC UCG UAG CAU UGU GCC UGU CUC CAG GUA CCA AGG AGA UAG (SEQ ID NO: 6)

[0074] 7. L

[0075] GCA CAG UGC UAU GAG UGU GCA CAG UGU CUG UGC GAC UGC ACC CAU CGC GAA AGG GUG UAG UCG CAU AGA CAU UGU GCU

[0076] GCA CAU UAG AGU CGC UGU AUG AGG GAU CGC GAA ACC CUC GUA

[0077] CAG CGG CUC UAG UGU GCU CGC GUG CCU CAG AGG ACC UGU CAC

[0078] CAG UCG ACA AGG UGA UAG GUC CUU UGA GGU ACG CGU CAC

[0079] UCG UAG CAU UGU GCC UGU CUC CAG UCG ACA AGG AGA UAG (SEQ ID NO: 7)

[0080] 8. AYFP

[0081] GGA UCC GGU UGC AAA GCA CAG UGC UAU GAG UGU GCA CGG GAU CCC GAC UGG CCG CAU CGC GAA AGU GGC CAG GUA ACG AAU GGA UCC UGU GCU GCA CAU UAG AGU CGC UGU AUG ACC CAU CGC GAA AGG GUC GUA CAG CGG CUC UAG UGU GCU CGC GUG CCU CAG AGG ACC UGU CAC CAU CGC GAA AGG UGA UAG

[0082] GUC CUU UGA GGU ACG CGU CAC UCG UAG CAU UGU GCC UGU CUC CAU CGC GAA AGG AGA UAG (SEQ ID NO: 8)

[0083] 9. YFPapt

[0084] GGC AAC CGG AUC CAA AGC AGC UUC UGG ACU GCG AUG GGA GCA CGA AAC GUC GUG GCG CAA UUG GGU GGG GAA AGU CCU UAA AAG AGG GCC ACC ACA GAA GCU GCA (SEQ ID NO: 9)

[0085] 10. BSTV

[0086] GCA CUA GAC CAC AAA GCA CAG UGC UAU GAG UGU CGC GAC GGA GAC GGU CGG GUC CAG AUA GGC CAG UCG ACA AGG UCU AUC UGU CGA GUA GAG UGU GGG CUC CGU CGC GUG CAC AUU AGA GUC GCU GUA UGC CAC AGU CGA CAA GUG GCG UAC AGC GGC UCU AGU GUG CUG CAC AGU GUC UGU GCG ACU GCA CCC AGU CGA CAA GGG UGU AGU CGC AUA GAC AUU GUG CUC ACU CGU AGC AUU GUG CCU GUC UCC AGU CGA CAA GGA GAU AG (SEQ

[0087] ID NO: 10)

[0088] 11. STVapt

[0089] GUG GUC UAG UGC UUU AUG CGG CCG CCG ACC AGA AUC AUG

[0090] CAA GUG CGU AAG AUA GUC GCG GGU CGG CGG CCG CAU (SEQ ID NO: 11)

[0091] REFERENCES

[0092] (1) Hyman, A. A., Weber, C. A. & J' ulicher, F. Liquid-liquid phase separation in biology. Annu. Rev. Cell Dev. Biol. 30, 39-58 (2014).

[0093] (2) Shin, Y. & Brangwynne, C. P. Liquid phase condensation in cell physiology and disease. Science 357 (2017).

[0094] (3) Lyon, A. S., Peeples, W. B. & Rosen, M. K. A framework for understanding the functions of biomolecular condensates across scales. Nat. Rev. Mol. Cell Biol. 22, 215— 235 (2021).

[0095] (4) Feric, M. et al. Coexisting liquid phases underlie nucleolar subcompartments. Cell 165, 1686-1697 (2016).

[0096] (5) Hirose, T., Ninomiya, K., Nakagawa, S. & Yamazaki, T. A guide to membraneless organelles and their various roles in gene regulation. Nat. Rev. Mol. Cell Biol. 24, 288- 304 (2023).

[0097] (6) Gomes, E. & Shorter, J. The molecular language of membraneless organelles. J. Biol. Chem. 294, 7115-7127 (2019).

[0098] (7) Molliex, A. et al. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163, 123-133 (2015).

[0099] (8) Patel, A. et al. A Liquid-to-Solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 162, 1066-1077 (2015). (9) Hastings, R. L. & Boeynaems, S. Designer condensates: A toolkit for the biomolecular architect. J. Mol. Biol. 433, 166837 (2021).

[0100] (10) Dai, Y., You, L. & Chilkoti, A. Engineering synthetic biomolecular condensates. Nature Reviews Bioengineering 1-15 (2023).

[0101] (11) Buddingh’, B. C. & van Hest, J. C. M. Artificial cells: Synthetic compartments with life-like functionality and adaptivity. Acc. Chem. Res. 50, 769-777 (2017).

[0102] (12) Guo, H. et al. Spatial engineering of e. coli with addressable phase-separated RNAs. Cell 185, 3823-3837. e23 (2022).

[0103] (13) Seeman, N. C. & Sleiman, H. F. DNA nanotechnology. Nature Reviews Materials 3, 1-23 (2017).

[0104] (14) Geary, C., Rothemund, P. W. K. & Andersen, E. S. A single-stranded architecture for cotranscriptional folding of RNA nanostructures. Science 345, 799-804 (2014).

[0105] (15) McRae, E. K. S. et al. Structure, folding and flexibility of co-transcriptional RNA origami. Nat. Nanotechnol. (2023).

[0106] (16) Stewart, J. M. et al. Modular RNA motifs for orthogonal phase separated compartments. bioRxiv (submitted) (2023).

[0107] (17) Biffi, S. et al. Phase behavior and critical activated dynamics of limited-valence DNA nanostars. Proc. Natl. Acad. Sci. U. S. A. 110, 15633-15637 (2013).

[0108] (18) Sato, Y., Sakamoto, T. & Takinoue, M. Sequence-based engineering of dynamic functions of micrometer-sized DNA droplets. Sci Adv 6, eaba3471 (2020).

[0109] (19) Brady, R. A., Brooks, N. J., Cicuta, P. & Di Michele, L. Crystallization of amphiphilic DNA C-Stars. Nano Lett. 17, 3276-3281 (2017).

[0110] (20) Liu, D. et al. Branched kissing loops for the construction of diverse RNA homooligomeric nanostructures. Nat. Chem. 12, 249-259 (2020).

[0111] (21) Kolpashchikov, D. M. Binary malachite green aptamer for fluorescent detection of nucleic acids. J. Am. Chem. Soc. 127, 12442-12443 (2005).

[0112] (22) Yerramilli, V. S. & Kim, K. H. Labeling RNAs in live cells using malachite green aptamer scaffolds as fluorescent probes. ACS Synth. Biol. 7, 758-766 (2018). (23) Filonov, G. S., Moon, J. D., Svensen, N. & Jaffrey, S. R. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution. J. Am. Chem. Soc. 136, 16299-16308 (2014).

[0113] (24) Brady, R. A., Brooks, N. J., Foder'a, V., Cicuta, P. & Di Michele, L. Amphiphilic- DNA platform for the design of crystalline frameworks with programmable structure and functionality. J. Am. Chem. Soc. 140, 15384-15392 (2018).

[0114] (25) Saleh, O. A., Jeon, B.-J. & Liedl, T. Enzymatic degradation of liquid droplets of DNA is modulated near the phase boundary. Proc. Natl. Acad. Sci. U. S. A. 117, 16160— 16166 (2020).

[0115] (26) Chamberlin, M. & Ring, J. Characterization of t7-specific ribonucleic acid polymerase: I. general properties of the enzymatic reaction and the template specificity of the enzyme. J. Biol. Chem. 248, 2235-2244 (1973).

[0116] (27) Arnold, S. et al. Kinetic modeling and simulation of in vitro transcription by phage T7 RNA polymerase. Biotechnol. Bioeng. 72, 548-561 (2001).

[0117] (28) Akama, S., Yamamura, M. & Kigawa, T. A multiphysics model of in vitro transcription coupling enzymatic reaction and precipitation formation. Biophys. J. 102, 221-230 (2012).

[0118] (29) Agarwal, S., Osmanovic, D., Klocke, M. A. & Franco, E. The growth rate of DNA condensate droplets increases with the size of participating subunits. ACS Nano 16, 11842-11851 (2022).

[0119] (30) Han, K. Y., Leslie, B. J., Fei, J., Zhang, J. & Ha, T. Understanding the photophysics of the spinach-DFHBI RNA aptamer-fluorogen complex to improve live-cell RNA imaging. J. Am. Chem. Soc. 135, 19033-19038 (2013).

[0120] (31) Li, X., Kim, H., Litke, J. L., Wu, J. & Jaffrey, S. R. Fluorophore-Promoted RNA folding and photostability enables imaging of single Broccoli-Tagged mRNAs in live mammalian cells. Angew. Chem. Int. Ed Engl. 59, 4511-4518 (2020).

[0121] (32) Levitz, P. Off-lattice reconstruction of porous media: critical evaluation, geometrical confinement and molecular transport. Adv. Colloid Interface Sci. 76-77, 71-106 (1998). (33) Testard, V., Berthier, L. & Kob, W. Influence of the glass transition on the liquidgas spinodal decomposition. Phys. Rev. Lett. 106, 125702 (2011).

[0122] (34) Di Michele, L. et al. Multistep kinetic self-assembly of DNA-coated colloids. Nat. Commun. 4, 2007 (2013).

[0123] (35) Hansch, C., Leo, A. & Hoekman, D. Exploring QSAR: hydrophobic, electronic, and steric constants Vol. 2 (American Chemical Society Washington, DC, 1995).

[0124] (36) Franco, E. et al. Timing molecular motion and production with a synthetic transcriptional clock. Proc. Natl. Acad. Sci. U. S. A. 108, E784-93 (2011).

[0125] (37) Jeon, B.-J., Nguyen, D. T. & Saleh, O. A. Sequence-Controlled adhesion and microemulsification in a Two-Phase system of DNA liquid droplets. J. Phys. Chem. B 124, 8888-8895 (2020).

[0126] (38) Shui, B. et al. RNA aptamers that functionally interact with green fluorescent protein and its derivatives. Nucleic Acids Res. 40, e39 (2012).

[0127] (39) Leppek, K. & Stoecklin, G. An optimized streptavidin-binding RNA aptamer for purification of ribonucleoprotein complexes identifies novel ARE-binding proteins. Nucleic Acids Res. 42, el3 (2014).

[0128] (40) Yamazaki, T., Nakagawa, S. & Hirose, T. Architectural RNAs for membraneless nuclear body formation. Cold Spring Harb. Symp. Quant. Biol. 84, 227-237 (2019).

[0129] (41) Schuster, B. S. et al. Biomolecular condensates: Sequence determinants of phase separation, microstructural organization, enzymatic activity, and material properties. J. Phys. Chem. B 125, 3441-3451 (2021).

[0130] (42) Yewdall, N. A., Andr'e, A. A. M., Lu, T. & Spruijt, E. Coacervates as models of membraneless organelles. Curr. Opin. Colloid Interface Sci. 52, 101416 (2021).

[0131] (43) Brady, R. A., Kaufhold, W. T., Brooks, N. J., Foder'a, V. & Di Michele, L. Flexibility defines structure in crystals of amphiphilic DNA nanostars. J. Phys. Condens. Matter 31, 074003 (2019).

[0132] (44) Tanner, N. K. Ribozymes: the characteristics and properties of catalytic RNAs. FEMS Microbiol. Rev. 23, 257-275 (1999). All publications mentioned herein (e.g. those listed above) are incorporated by reference to disclose and describe aspects, methods and / or materials in connection with the cited publications. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

Claims

CLAIMS1. A method of making a synthetic RNA condensate molecule comprising: transcribing a DNA sequence selected to encode a RNA condensate polynucleotide sequence so that the synthetic RNA condensate molecule is formed, wherein the DNA is selected to transcribe a synthetic RNA condensate molecule that: comprises 100-300 nucleotides; comprises complementary sequences that allow the RNA polynucleotide to selfassemble so as to form 3-5 arm structures comprising a stem and a loop, wherein: the stem in said arm structures comprise at least 10 paired nucleotides (i.e. are coupled to a complementary stretch of at least 10 paired nucleotides also in the synthetic RNA condensate molecule); the loops in said arm structures comprise at least 6 nucleotides; the base of the stems (distal to the loop) in said arm structures comprise at least 1-3 unpaired linker nucleotides that couple at least 10 paired nucleotides in a first arm structure to at least 10 paired nucleotides in a second arm structure; and a first loop in the synthetic RNA condensate comprises at least 6 nucleotides selected to be complementary to at least 6 nucleotides in a second loop in a synthetic RNA condensate such that the at least 6 nucleotides in the first loop of a first synthetic RNA condensate molecule and the at least 6 molecules in a second loop of a second synthetic RNA condensate molecule couple together so that the synthetic RNA condensate molecules aggregate to form a RNA condensate.

2. The method of claim 1, wherein: the synthetic RNA condensate molecule comprises a RNA aptamer motif sequence; and / or the synthetic RNA condensate molecule comprises a polynucleotide sequence to which a RNA aptamer couples via base pairing.

3. The method of claim 3, wherein:the RNA aptamer motif sequence is disposed in a loop; and / or the synthetic RNA condensate molecule comprises a 5' single stranded overhang of at least 10 or 15 residues, to which a RNA aptamer connects via base pairing.

4. The method of claim 1, wherein the at least 6 nucleotides in the first loop comprise a HIV-type Kissing-Loop motif.

5. The method of claim 1, wherein: the first loop and the second loop are disposed in a single synthetic RNA condensate molecule; and the first loop and the second loop in the single synthetic RNA condensate molecule cannot couple together.

6. The method of claim 1, wherein the DNA is transcribed within a synthetic cell.

7. The method of claim 1, wherein the method forms a plurality of synthetic RNA condensate molecules by transcribing a plurality of DNA sequences selected to encode a plurality of RNA condensation sequences so that a plurality of synthetic RNA condensate molecules are formed.

8. The method of claim 1, wherein the DNA is a linear dsDNA and / or is transcribed using a purified mRNA polymerase.

9. The method of claim 1, wherein the RNA condensate molecule is coupled to a protein and / or a nucleic acid.

10. A composition of matter comprising at least one synthetic RNA condensate molecule made by the method of claim 1.

11. The composition of claim 10, further comprising a protein.

12. The composition of claim 11, wherein the protein is coupled to the synthetic RNA condensate molecule via a RNA aptamer.

13. The composition of claim 10, wherein the synthetic RNA condensate molecule is disposed within a synthetic cell.

14. A RNA condensate formed by a plurality of aggregated synthetic RNA condensate molecules made by the method of claim 1.

15. A method of making a RNA condensate comprising:(a) combining together a plurality of synthetic RNA condensate molecules selected to: comprise 100-300 nucleotides; comprise complementary sequences that allow the synthetic RNA condensate molecules to self-assemble so as to form 3-5 arm structures comprising a stem and a loop, wherein: the stem in said arm structures comprise at least 10 paired nucleotides (i.e. are coupled to a complementary stretch of at least 10 paired nucleotides also in the synthetic RNA condensate molecule); the loops in said arm structures comprise at least 6 nucleotides; the base of the stems (distal to the loop) in said arm structures comprise at least 1-3 unpaired linker nucleotides that couple the at least 10 paired nucleotides in a first arm structure to the at least 10 paired nucleotides in a second arm structure; and at least a first loop in said arm structures comprises at least 6 nucleotides selected to be complementary to at least 6 nucleotides in at least a second loop such that the at least 6 nucleotides in a first loop of a first synthetic RNA condensate moleculeand the at least 6 molecules in a second loop of a second synthetic RNA condensate molecule couple together so that the synthetic RNA condensate molecules aggregate to form a RNA condensate; wherein the plurality of synthetic RNA condensate molecules are combined under conditions selected to facilitate condensation; and(b) allowing the plurality of synthetic RNA condensate molecules to aggregate so that the RNA condensate is made.

16. The method of claim 15, wherein the plurality of synthetic RNA condensate molecules comprise a homogeneous population of polynucleotides.

17. The method of claim 15, wherein the plurality of synthetic RNA condensate molecules comprise a heterogeneous population of polynucleotides.

18. The method of claim 15, wherein the plurality of synthetic RNA condensate molecules are combined within a synthetic cell.

19. The method of claim 15, wherein the plurality of synthetic RNA condensate molecules are combined under physiological conditions.

20. The method of claim 15, wherein the plurality of synthetic RNA condensate molecules are further coupled to proteins and / or nucleic acids.

Images