RNA Condensate as a Versatile Platform for Improving Fluorogenic RNA Aptamer Properties and Cell Imaging

Journal of the American Chemical Society, Journal Year: 2024, Volume and Issue: 146(7), P. 4402 - 4411

Published: Feb. 8, 2024

Fluorogenic RNA aptamers are valuable tools for cell imaging, but they still suffer from shortcomings such as easy degradation, limited photostability, and low fluorescence enhancement. Molecular crowding conditions enable the stabilization of structure, promotion folding, improvement activity functional RNA. Based on artificial condensates, here we present a versatile platform to improve fluorogenic aptamer properties develop sensors target analyte imaging in living cells. Using CUG repeat general tag drive phase separation, various aptamer-based condensates (FLARE) were prepared. We show that molecular FLARE can enzymatic resistance, thermostability, binding affinity aptamers. Moreover, systems be modularly engineered into (FLARES), which demonstrate enhanced brightness sensitivity compared free dispersed homogeneous solution. This scalable design principle provides new insights property regulation cellular imaging.

Language: Английский

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Atomic resolution map of the solvent interactions driving SOD1 unfolding in CAPRIN1 condensates

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(35)

Published: Aug. 22, 2024

Biomolecules can be sequestered into membrane-less compartments, referred to as biomolecular condensates. Experimental and computational methods have helped define the physical-chemical properties of Less is known about how high macromolecule concentrations in condensed phases contribute “solvent” interactions that remodel free-energy landscape other condensate-resident proteins, altering thermally accessible conformations and, turn, modulating function. Here, we use solution NMR spectroscopy obtain atomic resolution insights between immature form superoxide dismutase 1 (SOD1), which mislocalize aggregate stress granules, RNA-binding protein CAPRIN1, a component granules. studies CAPRIN1:SOD1 interactions, focused on both unfolded folded SOD1 states mixed phase demixed CAPRIN1-based condensates, establish CAPRIN1 shifts folding equilibrium toward state through preferential with ensemble, little change structure conformation. Key contacts H80-H120 region are identified, well interaction sites near arginine-rich aromatic-rich regions CAPRIN1. Unfolding shown coupled aggregation, while more stable zinc-bound, dimeric less susceptible unfolding when solvated by Our work underscores impact condensate solvent environment conformational resident proteins supports hypothesis ALS mutations decrease metal binding or dimerization function drivers aggregation

Language: Английский

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The emerging role of ATP as a cosolute for biomolecular processes

Biological Chemistry, Journal Year: 2023, Volume and Issue: 404(10), P. 897 - 908

Published: Sept. 1, 2023

ATP is an important small molecule that appears at outstandingly high concentration within the cellular medium. Apart from its use as a source of energy and metabolite, there increasing evidence for functions cosolute biomolecular processes. Owned to solubilizing kosmotropic triphosphate hydrophobic adenine moieties, versatile can interact with biomolecules in various ways. We here three models categorize these interactions apply them review recent studies. focus on impact solubility, folding stability phase transitions. This leads us possible implications therapeutic interventions neurodegenerative diseases.

Language: Английский

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Adenosine Triphosphate: The Primordial Molecule That Controls Protein Homeostasis and Shapes the Genome–Proteome Interface

Biomolecules, Journal Year: 2024, Volume and Issue: 14(4), P. 500 - 500

Published: April 19, 2024

Adenosine triphosphate (ATP) acts as the universal energy currency that drives various biological processes, while nucleic acids function to store and transmit genetic information for all living organisms. Liquid-liquid phase separation (LLPS) represents common principle formation of membrane-less organelles (MLOs) composed proteins rich in intrinsically disordered regions (IDRs) acids. Currently, IDRs are well recognized facilitate LLPS through dynamic multivalent interactions, precise mechanisms by which ATP affect still remain elusive. This review summarizes recent NMR results on human FUS, TDP-43, viral nucleocapsid (N) protein SARS-CoV-2, modulated acids, revealing following: (1) binds folded domains overlapping with nucleic-acid-binding interfaces; (2) interplay biphasically modulate competitively binding pockets Arg/Lys within IDRs; (3) energy-independently induces folding highest efficiency known so far. As likely emerged prebiotic monomeric world, a pivotal mechanism concentrate compartmentalize rare molecules forming primordial cells, appears control homeostasis shape genome-proteome interfaces throughout evolutionary trajectory, from origins modern cells.

Language: Английский

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Small CAG Repeat RNA Forms a Duplex Structure with Sticky Ends That Promote RNA Condensation

Journal of the American Chemical Society, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 14, 2025

Biomolecular condensation lays the foundation of forming biologically important membraneless organelles, but abnormal processes are often associated with human diseases. Ribonucleic acid (RNA) plays a critical role in formation biomolecular condensates by mediating phase transition through its interactions proteins and other RNAs. However, physicochemical principles governing RNA transitions, especially for short RNAs, remain inadequately understood. Here, we report that small CAG repeat (sCAG) RNAs composed six to seven repeats, which pathogenic factors Huntington's disease, undergo vitro cells. Leveraging solution nuclear magnetic resonance spectroscopy advanced coarse-grained molecular dynamic simulations, reveal sCAG form duplex structures 3'-sticky ends, where GC stickers initiate intermolecular crosslinking promote condensates. Furthermore, demonstrate can cellular within speckles. Our work suggests be promoted specific structural motifs, reducing reliance on sequence length multivalence. This opens avenues exploring new functions designing novel biomaterials based condensation.

Language: Английский

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Non-monotonous Concentration Dependent Solvation of ATP Could Help to Rationalize Its Anomalous Impact on Various Biophysical Processes

The Journal of Physical Chemistry Letters, Journal Year: 2025, Volume and Issue: unknown, P. 4305 - 4314

Published: April 23, 2025

Adenosine triphosphate (ATP), one of the biologically most important molecules, offers certain anomalous behavior during folding and liquid-liquid phase separation proteins RNAs. ATP can act as a "biological hydrotrope", i.e., it solubilizes hydrophobic or other biomolecules. However, upon exceeding physiological concentration range (2-10 mM), aggregation RNAs is promoted, an effect that not understood yet. Here we present time-domain frequency-domain Terahertz (THz) spectroscopic investigation to understand solvation with varying in 2-15 mM. Both time frequency domain studies adenosine (Adn), sodium (TPP), elucidate both well moiety contribute nearly equal propensity towards structure at low concentrations. higher concentrations (>10 dominates, which leads more structured shell followed by slower relaxation dynamics. This due triphosphate-driven reduced amount water-exposed groups, revealed molecular dynamics simulations. These observations could lead understanding complex role different biological systems.

Language: Английский

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Atomic resolution map of the solvent interactions driving SOD1 unfolding in CAPRIN1 condensates

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: May 2, 2024

Abstract Biomolecules can be sequestered into membrane-less compartments, referred to as biomolecular condensates. Experimental and computational methods have helped define the physical-chemical properties of Less is known about how high macromolecule concentrations in condensed phases contribute “solvent” interactions that remodel free-energy landscape other condensate-resident proteins, altering thermally accessible conformations and, turn, modulating function. Here, we use solution Nuclear Magnetic Resonance (NMR) spectroscopy obtain atomic resolution insights between immature form superoxide dismutase 1 (SOD1), which mislocalize aggregate stress granules, RNA-binding protein CAPRIN1, a component granules. NMR studies CAPRIN1:SOD1, focused on both unfolded folded SOD1 states mixed phase de-mixed CAPRIN1-based condensates, establish CAPRIN1 shifts folding equilibrium towards state through preferential with ensemble, little change structure conformation. Key contacts H80-H120 region are identified, well interaction sites near arginine-rich aromatic-rich regions CAPRIN1. Unfolding shown coupled aggregation, while more stable zinc-bound, dimeric less susceptible unfolding when solvated by Our work underscores impact condensate solvent environment conformational resident proteins supports hypothesis ALS mutations decrease metal binding or dimerization function drivers aggregation Significance Statement Biomolecular condensates concentrate nucleic acids regulate perform key biological functions. Although material these well-studied, much understood dynamics within them affected concentration biomolecules. In this study used structural modulated inside formed reveals biases an provides why case, mature affected.

Language: Английский

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Salt-Dependent Self-Association of Trinucleotide Repeat RNA Sequences

The Journal of Physical Chemistry Letters, Journal Year: 2024, Volume and Issue: 15(14), P. 3820 - 3827

Published: April 1, 2024

Repeat RNA sequences self-associate to form condensates. Simulations of a coarse-grained single-interaction site model for (CAG)n (n = 30 and 31) show that the salt-dependent free energy gap, ΔGS, between ground (perfect hairpin) excited state (slipped hairpin (SH) with one CAG overhang) monomer even) is primary factor determines rates yield self-assembly. For odd n, (GS) state, which an SH, used predict self-association kinetics. As monovalent salt concentration, CS, increases, ΔGS GS increase, decreases dimer formation. In contrast, shuffled sequences, same length sequence composition as (CAG)31, larger, suppresses their propensities aggregate. Although demonstrated explicitly (CAG) polymers, finding inverse correlation gap aggregation general.

Language: Английский

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Stick-slip unfolding favors self-association of expanded HTT mRNA

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: Oct. 9, 2024

In Huntington's Disease (HD) and related disorders, expansion of CAG trinucleotide repeats produces a toxic gain function in affected neurons. Expanded huntingtin (expHTT) mRNA forms aggregates that sequester essential RNA binding proteins, dysregulating processing translation. The physical basis aggregation has been difficult to disentangle owing the heterogeneous structure repeats. Here, we probe folding unfolding pathways expHTT using single-molecule force spectroscopy. Whereas normal HTT mRNAs unfold reversibly cooperatively, with 20 or 40 slip unravel non-cooperatively at low tension. Slippage base pairs is punctuated by concerted rearrangement adjacent CCG trinucleotides, trapping partially folded structures readily pair another strand. We suggest conformational entropy repeats, combined stable pairs, creates stick-slip behavior explains propensity mRNA.

Language: Английский

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RNA ensembles from in vitro to in vivo: Toward predictive models of RNA cellular function

Current Opinion in Structural Biology, Journal Year: 2024, Volume and Issue: 89, P. 102915 - 102915

Published: Oct. 13, 2024

Deepening our understanding of RNA biology and accelerating development RNA-based therapeutics go hand-in-hand-both requiring a transition from qualitative descriptions structure to quantitative models capable predicting behaviors, static an ensemble view. Ensembles are determined their free energy landscapes, which define the relative populations conformational states energetic barriers separating them. Experimental determination ensembles over past decade has led powerful predictive behavior in vitro. It also been shown during this time that cellular environment redistributes ensembles, changing abundances functionally relevant conformers vitro contexts with subsequent functional consequences. However, recent studies have demonstrated testing built highly measurements function, aided by emerging computational methodologies, enables modelling activity biological discovery.

Language: Английский

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