key: cord-0857223-5ii25jil
authors: Tulsiyan, Kiran Devi; Jena, Subhrakant; González-Viegas, María; Kar, Rajiv K.; Biswal, Himansu S.
title: Structural Dynamics of RNA in the Presence of Choline Amino Acid Based Ionic Liquid: A Spectroscopic and Computational Outlook
date: 2021-09-15
journal: ACS Cent Sci
DOI: 10.1021/acscentsci.1c00768
sha: 0babfc8655f5eb6fba86d76facebb2ae1bf1c602
doc_id: 857223
cord_uid: 5ii25jil

[Image: see text] Ribonucleic acid (RNA) is exceedingly sensitive to degradation compared to DNA. The current protocol for storage of purified RNA requires freezing conditions below −20 °C. Recent advancements in biological chemistry have identified amino acid-based ionic liquids as suitable preservation media for RNA, even in the presence of degrading enzymes. However, the mechanistic insight into the interaction between ILs and RNA is unclear. To the best of our knowledge, no attempts are made so far to provide a molecular view. This work aims to establish a detailed understanding of how ILs enable structural stability to RNA sourced from Torula yeast. Herein, we manifest the hypothesis of multimodal binding of IL and its minimal perturbation to the macromolecular structure, with several spectroscopic techniques such as time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) aided with molecular dynamics at microsecond time scales. Relevant structural and thermodynamic details from biophysical experiments confirm that even long-term RNA preservation with ILs is a possible alternative devoid of any structural deformation. These results establish a unifying mechanism of how ILs are maintaining conformational integrity and thermal stability. The atomistic insights are transferable for their potential applications in drug delivery and biomaterials by considering the advantages of having maximum structural retention and minimum toxicity.

RNA is a natural biopolymer with profound applications in medicine, 1 molecular or cellular biology, 2,3 and nano/ biotechnology. 4 RNA is key to the central dogma of life in carrying the DNA blueprint and drives protein synthesis. 5, 6 Besides serving as a component of the translation machinery, RNA also catalyzes chemical reactions and is instrumental in understanding disease biology. 7 Notably, to study RNAassociated processes, both in vivo and in vitro, the major challenge is their structural integrity in solution samples. This fundamental knowledge comes from their molecular architecture associated with the base pairing, electrostatic interactions, hydration capacity, and near environment effect, which requires harnessing to maintain RNA integrity during storage. 8 Cold storage is the commonly adopted method for RNA storage, in which samples are either stored at −20°C to −80°C or in liquid nitrogen. 9 During the past decade, new techniques have been developed for storing RNA at room temperature. Fabre et al. have shown that drying RNA samples and stabilizers can be stored at room temperature by using stainless steel capsules. 10 Reports by Seelenfreund et al. suggest desiccation as an efficient alternative for the preservation of RNA. 11 Moreover, silica encapsulation technology has also been emerging as a reliable method for the storage of RNA. 12 However, these technologies are obstreperous in nature, which comes with a high cost of maintenance.

On the other hand, ionic liquids (ILs) have recently gained interest, particularly for their role as efficient solvents to store biopolymers, proteins, and enzymes. 13−18 ILs are molten salts with low volatility, high ionic conductivity, and nontoxicity-like unique properties. 19−23 They can act as green alternatives to organic solvents for drug delivery and biochemistry applications. 18 ,24−27 Furthermore, they are suitable for long-term storage and extraction of macromolecules. 15,28−30 The suitability of ILs for storing proteins and DNA is widely practiced since these macromolecules can retain their functionalities. 31−36 However, in the case of RNA, their high vulnerability to degradation with external factors like temperature, pH, and enzymes pose a difficult task to store them for an extended period. 37 The advancement in finding a nontoxic and biocompatible solvent for RNA also demonstrates that ILs could be adequate and effective substitutes. Mamajonav et al. demonstrated the structural stability of nucleic acids in deep eutectic solvents (DES) and room temperature ILs (RTILs). 38 Mazid et al. investigated the role of ILs containing choline dihydrogen phosphate in the stability of siRNA in the presence of RNase A. 39 This was also supported by Fister et al. through their work on the RNA isolation from viruses using imidazolium and ammonium-based ILs. 22 A report by Pedro et al. also illustrates that choline-based ILs are suitable for preservation of recombinant RNAs. 40 Quental et al. successfully used amino acid-based IL solvents to extract and preserve RNA. 41 Despite these findings, the principal mechanism that enables ILs suitable for RNA storage still remains underemphasized and unexplored. This work aims to traverse the mechanistic details through an assiduous analysis of spectroscopic measurements and theoretical modeling. This is the first study that provides confirmatory spectroscopic evidence concerning the mode of interaction between choline amino acid-based ILs (ChAAILs) and RNA, to the best of our knowledge. In general, RNA prefers the Aform in buffer whereas DNA commonly exists as the B-form. Nucleic acid structures are well-known to be perturbed due to external factors, including temperature and pressure, disturbing the structural conformation. While RNA is extremely sensitive to degrading enzyme (RNAase), it requires additional precautions and measures while handling compared to other nucleic acids. However, in the case of tRNA, the modification of nucleobases has a significant role in regulating the overall stability of the biomolecule. 42 We developed an initial hypothesis and gained atomistic details of such interaction using theoretical modeling techniques followed by classical simulations up to microsecond time scales. The spectroscopic attestation of RNA−IL interaction was demonstrated using various techniques such as steady-state absorption (UV−vis and CD) and emission, TCSPC integrated with femtosecondfluorescence up-conversion, and fluorescence correlation spectroscopy (FCS). Isothermal titration calorimetry experiments were also carried out to provide thermodynamic insights on RNA−ILs binding. RNA melting experiments were performed to get a comprehensive picture of the thermal stability of RNA in the presence of ILs.

This work aims to investigate the structural insights from the interaction pattern and dynamics between RNA and ILs. Our focus is to explore (i) the site preference of ILs onto the macromolecular structure of RNA, (ii) dominant forces (electrostatic and van der Waal forces) that stabilize the complexes, (iii) physical stability, and (iv) effect of ILs on structural dynamics. Herein, we have adopted a combined computational and experimental approach to achieve the objectives mentioned above.

Binding Site Preference of ILs onto RNA Macromolecules. Molecular docking is used to explore the binding possibilities with their site preference based on the energetic evaluation. It forms the basis of our preliminary investigation to interpret the putative site of binding for the ChAAILs ([Ch][Glu] and [Ch][Asp]) with RNA. Initial calculations reveal that Ch binding preference is mainly onto the minor groove of the RNA structure, as shown in Figure 1A ,B. However, there are no such site preferences for the counterion amino acids. The obtained binding energy of the docked complexes is ∼−4 kcal·mol −1 , irrespective of Glu and Asp. The orientation of the cholinium ion in the lowest energy conformation is stabilized through hydrogen bonding ( Figure  1 ). The O−H group of the ligand is involved in H-bonding with nucleobases (O−H···N). In the case of DNA, a previous study has shown that Lennard-Jones (LJ) potential between ILs and DNA contributes to −10 to −23 kcal·mol −1 to the overall stabilization energy. However, our results for RNA show the LJ contribution is minimum (−1 to −2 kcal·mol −1 ), indicating that there is feeble hydrophobic interaction between the ILs and RNA. Furthermore, we compared our docking results with the ethidium bromide (EB) molecule, which is also known to bind preferably at the minor-groove of nucleic acid structures, as shown in Figure 1C . Indeed, the lower energy conformation obtained also shows preference to the minorgroove location, like Ch, which further triggered us to verify this experimentally.

Binding Mode Analysis with Absorption and Fluorescence Spectroscopy. UV−vis absorbance measurement is valuable for determining RNA purity and confirming the binding specificity of ILs. Since small molecule binding to RNA is possible through three modes, viz., intercalation, groove binding, or external binding, the change in absorbance and a shift in absorption maxima (λ max ) upon binding are good indicators. 43 33 The intercalation mode of EB binding to RNA was confirmed from the hypochromic and bathochromic shift ( Figure S5A ), which is in line with the previous report. 44 Further addition of IL to the RNA−EB complex resulted in hyperchromism at 280 nm, confirming the interactions present between RNA and ILs, whereas no spectral shift for the peak at 480 nm (which corresponds to the EB probe) attributes to the absence of interactions among ILs and EB. No complexation of EB with ILs was further confirmed from the minimal change in the absorption spectra of EB on the addition of ILs in the absence of RNA ( Figure  S5B ). Similar observations are made with other fluorescent probes 4′,6-diamidino-2-phenylindole (DAPI) ( Figure  S5C ,D).

As the RNAs have low fluorescence quantum yields, the fluorescence probes such as EB and DAPI are ideal and 35 Another study by Dipak et al. on dye displacement also affirms that [Ch] can preferentially displace EB as well as DAPI from DNA. 33 Though the interaction between ChAAILs and RNA happens in a shorter period (picosecond or nanosecond time scale regime), it is critical to get more insights on structural dynamics using the time-resolved fluorescence. Figure 2E Thermodynamic Behavior of RNA. The thermodynamic behavior of RNA in the presence of ILs was analyzed using ITC. First, we titrated RNA against EB to determine its binding constant (K b ), which was found to be 4 × 10 4 mol −1 ( Figure S7A ). Next, the binding affinities of ILs with RNA were calculated by the same method. A higher concentration of ILs was required, owing to its smaller ionic size. The integrated heat data of RNA in the absence and presence of ILs with a single binding site model fitting (χ 2 = 0.97) are provided in the Supporting Information (Figure S7B,C) . Figure 3 . The conformational dynamicity of RNA achieves states of equilibrium at the 0.3 μs time scale. Although RMSD shows a straight line (deviation <2 Å), it is necessary to compare the dynamic pattern of RNA in the absence of ILs. 46 A control simulation of RNA alone with an equivalent time scale shows that equilibration was achieved at the <100 ns time scale ( Figure 3A,B) . Furthermore, the deviation in the remaining time scale is comparable to that in the presence of ILs.

The binding preferences of cholinium cations are primarily in the minor groove region of RNA; however, we observed multiple (heterogeneity) binding poses. Different modes of [Ch] onto the RNA macromolecule can be visualized in Figure  3C ,D. For the natural and modified nucleobase of RNA, we refer the readers to PDB code1EHZ. Additionally, to determine the global structural changes among the three simulation systems, RMSDs and interaction network fidelity (INF) are taken as a reference to calculate the deformation index (DI). 47 The mentioned parameters help to analyze the structural stability and packing of nucleic acid structures from the simulation snapshots. 48 A strong emphasis on these parameters in the context of three-dimensional RNA structures was discussed earlier by Parisien et al. 47 INF describes the conservation of base−base pairing and the stacking interactions. Identical conservation in successive snapshot scores as 1, whereas no conservation scores 0. In this concern, Kuḧrovaé t al. reported INF as a reliable tool to determine the correct folding of RNA structures rather than RMSD. 49 The DI parameter accounts for both the RMSD and INF to evaluate the ratio between the structural dynamics and base-pair interaction. Figure Conformational Stability and Regeneration Study. Fluorescence correlation spectroscopic (FCS) studies were carried out to investigate the structural integrity of RNA in the presence of IL. FCS provides information about the hydrodynamic radius and diffusion time of the biomacromolecules. It is known that no significant perturbation tends to occur in DAPI-bound DNA structures in the presence of ILs. 35 In this study, we collected FCS data for DAPI and DAPI−RNA samples (see the methodology in the Supporting Information). The hydrodynamic radius (R H ) of RNA alone is estimated to be 7 ± 1 nm, which is in line with the reported data. 50 Figure S8A −C. The experimental measurements can also be correlated using the gyration radius from theoretical calculations ( Figure S9) .

Analysis of the thermodynamic stability of RNA−IL binding was undertaken with the UV melting measurements. 53, 54 Here, the increase in temperature is responsible for the release of stacking between nucleobases. It results in a strong-hyperchromic shift in absorbance spectra. The melting curves for RNA alone and in the presence of ChAAILs for absorbance at 260 nm are shown in Figure 4B . The melting temperature (T m ) of RNA is found to be 65°C, which agrees with the reported literature. 53 The addition of ChAAILs leads to an increase in the T m by 5°C. The increase in T m of RNA in the presence of ChAAILs indicates that RNA is more stable in the presence of ILs, which follows the previous reports by Pedro et al. 40 They showed that T m of sRNA increases by almost 9°C in the presence of Ch[DHP]IL, thus providing additional stability to RNA. 40 Figure 3H ). Such confirmation for the long-term stability of nucleic acid structures in the presence of Ch and imidazolium ILs binding with DNA is also reported based on MD simulations. 32 This increment in T m is attributed to the fact that ILs add stability to the secondary structure of RNA. However, it also indicates the possibility of multiple binding poses because a single preference for groove binding cannot bring additional stabilization. Such observations are in line with the properties of DNA in the presence of ILs. 33 The assessment of dynamic insights and structural perturbations is possible using CD spectroscopy. It is a wellknown fact that the secondary structure of RNA has A-type double-stranded helices. 55 It gives a characteristic positive peak at 270 nm with a molar ellipticity of 9 mdeg and two negative peaks at 241 and 210 nm with a corresponding molar ellipticity of −2.5 and −5 mdeg, respectively. CD measurements of our sample with an increasing concentration of ChAAILs are shown in Figure 4C ,D. No substantial changes occur in both the positive and negative peaks of RNA upon binding to ILs. It remains constant even after adding 500 mM IL, which provides additional validity that RNA structures retain their structural stability in ChAAILs. The minimal change in CD spectra in the presence of ILs is in good agreement with the previous reports, 33, 40 where it is observed that ILs do not alter the conformation of nucleic acids. Using the same method, we further compare the CD spectra of stored and extracted RNA from the IL solution (after 3 weeks). Figure 4E shows that there is no change in the confirmation of extracted RNA.

The absorption spectra of the RNA sample extracted from the IL solution are shown in Figure 4F . The ratio between the absorbance at 260 and 280 nm confirms the stable conformation of the stored sample. These observations prove that no structural degradation or significant perturbation to the three-dimensional structure of the RNA happens in the solution containing ILs. This is also in good agreement with the observations made from the MD trajectories, with no Figure 3 ) with and without ILs. RNA−IL Binding Is Driven by Electrostatic Interaction. For additional insights into structural dynamics, we analuzed MD trajectories carefully. From the RMSD and basestacking RNA analysis, no evidence is available to indicate any significant structural perturbations. The analyzed binding modes of Ch determine the proximal distance (min) between any atom of RNA and ligand ( Figure 5) , where min <0.4 nm represents a very close interaction between Ch and RNA. However, the fluctuation in the min distance denotes that ILs are interacting in several instances in the simulation. More evidence of binding between Ch and RNA is available through the number of hydrogen bonds. These hydrogen bonds are mostly forming between the positively charged Ch and the negatively charged phosphate group. These data also indicate a lower min value in simulation time because of one or more Hbonds. The water molecules present in the close shell of Ch also mediate the multiple binding poses with RNA. These observations are in good agreement with the experimental measurements. Further, we compared these data with the behavior of IL binding to DNA. In the case of RNA, we found that the binding is comparatively weak, as indicated by the binding free energy, H-bonds, and the r-min distance.

The energetic contribution analysis focuses on the nonbonded interaction between Ch and the rest of the system (RNA, water molecules, and counterions). The hydrophobic contribution (Lennard−Jones) in the Ch dynamics is negligible and appears as ∼−3 kcal·mol −1 . However, the driving force in these simulations is from the electrostatic interaction. Such energetic contribution accounts for ∼−35 kcal·mol −1 . This is obvious because the Ch binding is mainly through the H-bonding. Thus, we conclude that IL binding with RNA is not mediated through the surface-bound state but the groove-bound state. At the same time, the critical stabilization force is not through the hydrophobic and van der Waals forces. Our simulation results also confirm that the amino acid counterpart imparts no significant difference in the overall behavior. These observations agree with the observed spectral features from UV−vis, fluorescence, and CD spectroscopy.

Transferability of ILs−RNA Interaction for Potential Application. ILs are valuable alternatives compared to organic solvents due to their superior physiochemical properties like low toxicity, low chemical reactivity, high thermal stability, low inflammability, and high conductivity. Ch has been recognized as central to ILs due to the fact they are less cytotoxic, biodegradable, and easily synthesizable based on green chemistry. 56 On the other hand, a wide variety of anionic counterparts of ILs with Ch has been reported in conjunction with nucleic acids including chlorides, acesulfamates, and bis(trifluoromethylsulfonyl)imides; 57 imidazolium-based; 58 morpholinium-based; 35 pyridinium-based; 59 glycolate and pyruvate-based; 60 and glycolateacetate and dihydrogen phosphate. 61 Though all of these anionic counterparts have reported multimodal binding, they are not acceptable for routine use of ILs either due to expensive chemical route of synthesis, differential chemical stability, or being nonbiodegradable. Furthermore, the level of cytotoxicity is different, which otherwise is higher compared to AAILs. 62 Note that long-term storage of RNA with marked extract efficacy is challenging and an evolving research field. Other than biochemical use, RNA also has potential pharmaceutical applications. For example, RNA robed with ammonium-based ILs are useful prodrugs for dermatological treatment. 63 It is evidenced that topical delivery of RNA using ammoniumbased ILs have remarkable therapeutic efficacy. 64 Hence, ILs can also be useful drug delivery agents for RNA. Since ILs facilitate additional stability to RNA, they also can trigger a new direction for their use in the pharmaceutical and biotechnology research.

Besides ILs are salts with organic cations or anions, having irregular structure to delocalize their charges. The chemical shape of these molecules and low charges allow these molecules to (i) avoid tight packing during crystallization and (ii) acquire property of low melting points. Considering the amino acid-based ILs (AAILs), the important factor determining their efficacy is the polarity. Since this property determines the solubility of solute, miscibility with other solvents, and its reaction efficiency as a solvent, the polarizability of AAILs must be compared for their usefulness in specific applications. According to the Kamlet−Taft parameters for a series of AAILs, the polarizability ranges from minimum 1.04 [Val] to maximum 1. 13 [Asp] and [Glu] . 65 Thus, we hypothesized that AAILs irrespective of the amino acid counterpart could display identical behavior when used as a solvent medium for RNA.

The current work highlights the detailed mechanism responsible for the long-term storage of RNA by using ILsbased solvent medium. Herein, we elucidate the molecularlevel understanding of the structural perturbation of RNA induced through choline amino acid-based ILs. The initial hypothesis for structural factors driving the interaction between ILs and RNA is obtained using computational modeling. The experimental validation of these findings is possible with suitable techniques such as steady-state fluorescence, UV−vis absorbance, circular dichroism spectroscopy, and isothermal titration calorimetry (ITC). Fluorescence correlation spectroscopy confirms that there is no change in the hydrodynamic radius of RNA in the presence of ILs, revealing the structural integrity of the biomolecule in the IL environment, which is further confirmed by circular dichroism analysis. UV−vis spectra analysis indicates multimodal binding of CAAILs with RNA, whereas dye displacement studies through fluorescence emission confirm IL molecules' intrusion into the minor groove of RNA.

Interestingly, we found that increases in IL concentration do not affect structural instability. From isothermal titration calorimetry and molecular docking, the binding energy of ILs with RNA is ∼−4 kcal·mol −1 , which indicates weak binding of ILs to RNA. Our results demonstrate that ILs preserve the structural integrity of RNA and are useful as nontoxic and biocompatible solvent. The lower binding energy of ILs with RNA further accounts for RNA extraction from the IL media. The significance of this study lies in the spectroscopic and theoretical evidence that noncytotoxic and eco-friendly ChAAILs are useful for the extraction and safe storage of RNA. The molecular-level information obtained from the present results can be valuable guidance for designing suitable ILs for the in vivo application of RNA. 

Kar − Faculty II-Mathematics and Natural Sciences

Homi Bhabha National Institute

Homi Bhabha National Institute

Homi Bhabha National Institute

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