G-Clamp Heterocycle Modification Containing Interstrand Photo-Cross-Linker to Capture Intracellular MicroRNA Targets.

MicroRNAs (miRNAs) play indispensable roles in post-transcriptional gene regulation. The identification of target mRNAs is essential for dissecting the recognition basis, dynamics, and regulatory mechanism of miRNA-mRNA interactions. However, the lack of an unbiased method for detecting weak miRNA-mRNA interactions remains a long-standing obstacle for miRNA research. Here, we develop and provide proof-of-concept evidence demonstrating a chemical G-clamp-enhanced photo-cross-linking strategy for covalent capture of intracellular miRNA targets in different cell lines. This approach relies on an aryl-diazirine-G-clamp-modified-nucleoside (ARAGON) miRNA probe containing an alkynyl group that improves the thermal stability of miRNA-target mRNA duplex molecules and can rapidly cross-link with the complementary strand upon UV 365 nm activation, enhancing the transient capture of mRNA targets. After validating the accuracy and binding properties of ARAGON-based miRNA probes through the successful enrichment for the known targets of miR-106a, miR-21, and miR-101, we then extend ARAGON's application to screen for previously unknown targets of different miRNAs in various cell lines. Ultimately, results in this study uncover GAB1 as a target of miR-101 in H1299 lung cancer cells and show that miR-101 silencing of GAB1 can promote apoptosis in H1299 cells, suggesting an oncogenic mechanism of GAB1. This study thus provides a powerful and versatile tool for enhanced screening of global miRNA targets in cells to facilitate investigations of miRNA functions in fundamental cellular processes and disease pathogenesis.

Liquid-liquid phase equilibrium and the effect of a water-soluble polymer on the interaction between droplets in water-in-oil microemulsions.

The liquid-liquid phase equilibria of {water/PEG200/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/n-decane} with the molar ratio of water to AOT being 37.9 and various concentrations cPEG of PEG in water were measured in this study. The critical exponent ß corresponding to the width of the coexistence curve was determined, which showed good agreement with the 3D-Ising value and supported the proposal of the pseudo binary droplet solution for these multiple microemulsions. A previously developed thermodynamic approach based on the Carnahan-Starling-van der Waals type equation was improved and used to analyze the coexistence curve data of {water/PEG200/AOT/n-decane} microemulsions to deduce the interaction properties between droplets and further to investigate the effect of the additive PEG200 on these interactions. It was found that the addition of PEG200 into the {water/AOT/n-decane} microemulsion resulted in the decrease in the critical temperature and the interaction enthalpy and entropy. Both the interaction enthalpy and entropy decreased first, then increased with an increase of cPEG and had minimum values at cPEG = 25 g L-1, which showed the same tendency as the isothermal titration microcalorimetric results for the {water/PEG200/AOT/isooctane} microemulsion we reported very recently. The effects of the interaction enthalpy and the entropy on the phase separation and their dependences on cPEG were discussed and related to the effects of the additive on the rigidity of the interface layer of the microemulion droplet.

The heat capacities and critical behaviors of binary ionic solutions.

The heat capacities of nine binary room temperature ionic solutions {[C4mim][BF4] + 1,2-butandiol}, {[C8mim][BF4] + 1-pentanol}, {[C8mim][BF4] + 2-pentanol}, {[C8mim][BF4] + 1-hexanol}, {[C8mim][BF4] + 1-heptanol}, {[C8mim][PF6] + 1-propanol}, {[C8mim][PF6] + 1-butanol}, {[C8mim][PF6] + 2-butanol} and {[C8mim][PF6] + tert-butanol} are reported herein. The combination of the data obtained with the corresponding measured coexistence curves infers that the critical asymmetry parameter of the coexistence curves linearly varies with the molar volume ratio of the two components for each of the studied binary ionic solutions after the heat capacity contribution is considered. This indicates the importance of the heat capacity contribution to the critical asymmetry. For further analysis of the critical characteristics of the ionic solutions, a large amount of experimental data was collected and systematically discussed in detail regarding which critical character is important in the binary ionic solutions. A general increasing tendency for the RPM (restricted primitive model)-rescaled critical parameters with the relative permittivity εr,c of the solvent at the critical temperature of the corresponding system was found. This is attributed not only to the screening effect of the solvent medium, but also to solvophobic interactions, which both increase with εr,c. This study also demonstrates that the critical amplitudes increased, while the relative contribution of the heat capacity to the asymmetry of the coexistence curve decreased with an increase in εr,c.

The inter-micellar interaction enthalpies of DTAB/TX100 mixed micelles and their structural transitions.

The heats of mixing for a series of DTAB/TX100 mixed surfactant aqueous solutions were measured by flow-mixing calorimetry and isothermal titration calorimetry (ITC) at 298.15 K and 85 kPa, which were used to calculate the inter-micellar interaction enthalpies (-ΔHC). The signs of -ΔHC for pure DTAB and TX100 micellar systems were contrary to the inter-micellar interaction parameters reported for the same systems in the literature, suggesting that these interaction parameters might have a Gibbs free energy character dominated by entropy changes. It was found that the inter-micellar interaction enthalpies varied with the total surfactant concentration and the mixed ratio of the two surfactants, and characterized different structures of the mixed micelles. These phenomena were discussed based on the effects of structural water around the micellar interface, the hydration of counterions, and the repulsive Coulombic interaction.

Critical universality and asymmetry of ionic solution {iodobenzene + 1-decyl-3-methyl-imidazolium bis(trifluorosulfonyl)imide}.

The liquid-liquid coexistence curve, heat capacity in the critical and noncritical regions, and the turbidity in critical one-phase and two-phase regions of the binary solution {iodobenzene + 1-decyl-3-methylimidazolium bis(trifluorosulfonyl)imide ([C10mim][NTf2])} have been precisely measured. From the data collected in the critical region, the critical exponents α, ß, γ and ν, as well as the universal critical amplitude ratios RB and X were obtained and were shown to agree well with their theoretical values for the 3D-Ising universality class, which further confirmed the 3D-Ising criticality of ionic solutions even in a very low relative permittivity solvent. The coulombic character of the studied system was suggested by the small values of the RPM reduced critical temperature and density. Furthermore, it was found that both the asymmetric behaviors of the diameter of the coexistence curve and the osmotic compressibility could be well described using the complete scaling theory.

Liquid-Liquid Phase Equilibria and Interactions between Droplets in Water-in-Oil Microemulsions.

The liquid-liquid phase equilibria of [water/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/n-decane] with the molar ratio w0 of water to AOT being 37.9 and [water/AOT/ethoxylated-2,5,8,11-tetramethyl-6-dodecyne-5,8-diol(Dynol-604)/n-decane] with w0 = 37.9 and the mole fraction α of Dynol-604 in the total surfactants being 0.158 were measured in this study. From the data collected in the critical region, the critical exponent ß corresponding to the width of the coexistence curve was determined, which showed good agreement with the 3D-Ising value. A thermodynamic approach based on the Carnahan-Starling-van der Waals type equation was proposed to describe the coexistence curves and to deduce the interaction properties between droplets in the microemulsions. The interaction enthalpies were found to be positive for the studied systems, which evidenced that the entropy effect dominated the phase separations as the temperature increased. The addition of Dynol-604 into the (water/AOT/n-decane) microemulsion resulted in the decrease in the critical temperature and the interaction enthalpy. Combining the liquid-liquid equilibrium data for (water/AOT/n-decane) microemulsions with various w0 values determined previously, it was shown that the interaction enthalpy decreased with w0 and tended to change its sign at low w0, which coincided with the results from the isothermal titration calorimetry investigation. All of these behaviors were interpreted by the effects of entropy and enthalpy and their competition, which resulted from the release of solvent molecules entrapped in the interface of microemulsion droplets and were dependent on the rigidity of the surfactant layers and the size of the droplet.

Structural rearrangement in the aqueous solution of surface active ionic liquid 1-butyl-3-methylimidazolium bis(2-ethylhexyl) sulfosuccinate.

The aggregation behaviors of surface active ionic liquid 1-butyl-3-methylimidazolium bis(2-ethylhexyl) sulfosuccinate in aqueous solutions were investigated by conductometry, densimetry, fluorimetry, (1)H nuclear magnetic resonance ((1)H NMR) spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM), which confirmed two distinguished critical concentrations. The first critical concentration was believed to be the critical aggregation concentration (CAC), where two different types of aggregates were formed, namely, micelles with the size of 10 nm and vesicles with the size of 100 nm. The second critical concentration at two-fold CAC was suggested to be resulting from the insertion of the imidazolium cations into aggregates.

Effects of water content and chain length of n-alkane on the interaction enthalpy between the droplets in water/sodium bis(2-ethylhexyl)-sulfosuccinate/n-alkane microemulsions.

The concentration-dependent enthalpies of mixing for water/sodium bis(2-ethylhexyl)-sulfosuccinate (AOT)/n-alkane microemulsions with different water contents ω0 and chain lengths n of n-alkane were determined by isothermal titration microcalorimetry (ITC) and flow-mixing microcalorimetry at 298.15 K and used to calculate the interaction enthalpies (-ΔH(C)) between the droplets. It was found that -ΔH(C) increased with ω0, and changed from negative to positive at about ω0 = 10. The investigation of the dependence of -ΔH(C) on n revealed that the values of -ΔH(C) were negative and had a minimum for ω0 = 5; while they were positive and had a maximum for ω0 = 15. These phenomena were discussed based on the competition of the overlapping contribution of the surfactant tails between two neighbouring droplets and the penetration contribution of the solvent molecules into the surfactant tails. These results indicated the important role of entropy in the stability of the microemulsion systems.

Critical Anomalies of Two SN1 Hydrolysis Reactions in Critical Binary Solutions.

Rates of SN1 hydrolysis reactions for 2-chloro-2-methylbutane in the critical solution of isobutyric acid + water and for 2-bromo-2-methylpropane in the critical solution of triethylamine + water in the one-phase region and at various temperatures have been determined respectively by conductance measurements. It was found that the reaction rates at different temperatures for those two SN1 reactions were well described by the Arrhenius equation in the noncritical region, whereas near the critical points the critical slowing down was clearly detected. These results are inconsistent with a previous report in the literature (J. Phys. Chem. A 2003, 107, 8435 - 8443). Reanalyzing the literature data, we found that if an Arrhenius equation appropriate to either the one-phase region or the two-phase region being examined was used as the background, a critical slowing down rather than a speeding up was detected for the reaction in both the one-phase region and the two-phase region. The experimental data from different sources were fitted with a simplified crossover formalism characterizing the critical effect on the reactions to determine the critical slowing down exponents, which were found to be about 0.04, showing that only a dynamic critical slowing down could be detected for these reaction systems. This phenomenon was attributed to the fact that the SN1 hydrolysis reaction is neither a first-order nor a pseudo-first-order reaction in the reverse direction and the kinetic measurements were carried out in a region quite far from the equilibrium of the reactions.

Volumetric properties of water/AOT/isooctane microemulsions.

The densities of AOT/isooctane micelles and water/AOT/isooctane microemulsions with the molar ratios R of water to AOT being 2, 8, 10, 12, 16, 18, 20, 25, 30, and 40 were measured at 303.15 K. The apparent specific volumes of AOT and the quasi-component water/AOT at various concentrations were calculated and used to estimate the volumetric properties of AOT and water in the droplets and in the continuous oil phase, to discuss the interaction between the droplets, and to determine the critical micelle concentration and the critical microemulsion concentrations. A thermodynamic model was proposed to analysis the stability boundary of the microemulsion droplets, which confirms the maximum value of R being about 65 for the stable AOT/water/isooctane microemulsion droplets.

The interactions of ι-carrageenan with cationic surfactants in aqueous solutions.

The interactions between the anionic polymer ι-carrageenan (IC) and the cationic surfactants 1-dodecyl-3-methylimidazolium bromide (C12mimBr), dodecyltrimethylammonium bromide (DTAB) and ethyl-α,ω-bis(dodecyldimethylammonium)dibromide (12-2-12) have been studied by fluorimetry and isothermal titration calorimetry. Our experimental results showed that at a low surfactant concentration, the monomers adsorbed on the IC chains through the electrostatic attraction, followed by the formation of induced micelles on the IC chains through the hydrophobic interaction until the IC chains are saturated by surfactant molecules; after that the added surfactant formed free micelles in the solution. A pseudo-phase-equilibrium thermodynamic model was proposed to explain the experimental results and to understand the mechanisms of the interactions in these three systems. Moreover, the salt effect on the interactions was investigated and found that it changed the critical concentrations but not the interaction mechanism.

Solvent dependent interactions between droplets in water-in-oil microemulsions.

In this paper, we investigated the dilution enthalpies of the droplets in water/AOT/oil microemulsions with oil being isooctane, decane, or cyclohexane by isothermal titration microcalorimetry (ITC). Combining with the results obtained from the study of the water/AOT/toluene system in our previous work, it was found that the enthalpy interactions between droplets for isooctane and decane systems were repulsive, while the enthalpy interactions were attractive for cyclohexane and toluene systems. The repulsive droplet interaction for the isooctane system was also confirmed by static light scattering. The solvents appear to play important roles in varying the droplet enthalpy interactions from positive to negative, and the entropy contribution seems to be dominant for the stability of these microemulsion droplet systems.

Asymmetric criticality of binary ionic solutions containing 1-butyl-3-methylimidazolium tetrafluoroborate and alcohol.

The liquid-liquid coexistence curves for binary solutions {1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim][BF4]) + 1-propanol} and {1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim][BF4]) + 2-propanol} have been precisely measured. The values of the critical exponents ß obtained from the liquid-liquid equilibrium data in the critical region confirmed the 3D-Ising universality. The isobaric heat capacities per unit volume were measured for {[C4mim][BF4] + 1-propanol (or 2-propanol, 1,3-propanediol, 1,4-butanediol)} in both critical and non-critical regions. The experimental results indicate a major solvophobic contribution to the criticality for the studied ionic solutions. The complete scaling theory was applied to well represent the asymmetric behavior of the diameter of the coexistence curves with the consideration of the heat capacity contribution. It was found that the contribution of the heat capacity related term in the ionic solution decreased with the increase of the permittivity of alcohol and was more important in the description of the asymmetry of the coexistence curve of the ionic solutions than that of the molecular solutions.

Kinetics of the oxidation of iodide ion by persulfate ion in the critical water/bis(2-ethylhexyl) sodium sulfosuccinate/n-decane microemulsions.

In this work, we studied the kinetics of the oxidation of iodide ion by persulfate ion in the critical water/bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/n-decane microemulsions with the molar ratios of water to AOT being 35.0 and 40.8 via the microcalorimetry at various temperatures. It was found that the Arrhenius equation was valid for correlating experimental measurements in the noncritical region, but the slowing down effect existed significantly in the near critical region. We determined the values of the critical slowing down exponent and found it to be 0.187 ± 0.023 and 0.193 ± 0.032, respectively, which agreed well with the theoretical value of 0.207 predicted by the Griffiths-Wheeler rule for the singularity of the dimer/monomer droplet equilibrium in the critical AOT/water/n-decane microemulsions.

GLORI for absolute quantification of transcriptome-wide m6A at single-base resolution.

N6-methyladenosine (m6A) is the most abundant posttranscriptional chemical modification in mRNA, involved in regulating various physiological and pathological processes throughout mRNA metabolism. Recently, we developed GLORI, a sequencing method that enables the production of a globally absolute-quantitative m6A map at single-base resolution. Our technique utilizes the glyoxal- and nitrite-based chemical reaction, which selectively deaminates unmethylated adenosines while leaving m6A intact. The RNA library can then be prepared using a modified library construction protocol from enhanced UV crosslinking and immunoprecipitation (eCLIP) or commercial kits. Here we provide a detailed protocol for proper RNA sample handling and provide further guidelines for the use of a tailored bioinformatics pipeline (GLORI-tools) for subsequent data analysis. Compared with current methods, this new method is both exceptionally sensitive and robust, capable of identifying ~80,000 m6A sites with 50 Gb sequencing data in mammalian cells. It also provides a quantitative readout for m6A sites at single-base resolution. We hope the technique will provide a precise and unbiased tool for investigating m6A biology across various fields. Basic expertise in molecular biology and knowledge of bioinformatics are required for the protocol. The entire procedure can be completed within a week, with the library construction process taking ~4 d.

Kinetics of the reaction of crystal violet with hydroxide ion in the critical solution of 2-butoxyethanol + water.

The kinetics of alkaline fading of crystal violet (CV) has been studied by UV spectrophotometry and microcalorimetry in the critical binary solution of 2-butoxyethanol + water at the initial reaction stage and various temperatures. It was found that the first-order rate constants obtained from these two methods are well accorded with each other, and the temperature dependence of the rate constant obeyed the Arrhenius equation in a temperature region far from the critical point. The critical slowing down was detected by both methods near the critical point. A simple empirical crossover model was proposed and used to analyze the experimental data to obtain the critical exponents, which were 0.158 ± 0.013 and 0.133 ± 0.012 from UV spectrophotometry and microcalorimetry, respectively, and the former was in good agreement with the theoretical prediction of 0.151. The slight lower value derived from microcalorimetry was attributed to the stirring in the microcalorimeter, which weakened the critical reduction of the diffusion coefficient.

Asymmetric criticality of the osmotic compressibility in binary mixtures.

Heat capacities in the critical and the non-critical regions for {benzonitrile + tridecane} and {benzonitrile + pentadecane}, and light scattering for {benzonitrile + undecane}, {benzonitrile + dodecane}, {benzonitrile + tridecane}, {benzonitrile + tetradecane}, {benzonitrile + pentadecane}, and {benzonitrile + hexadecane} in the critical two-phase region were measured. Light scattering measurements confirmed the existence of the asymmetry for the osmotic compressibility while no such asymmetry was observed for the correlation length. An analysis of the osmotic compressibility asymmetry suggested the dominance of the singular term |ΔT[circumflex]|(ß), which supports the complete scaling theory. The consistency of the complete scaling theory in descriptions of different asymmetry behaviors was also discussed. Moreover, it was found that the contribution of the heat capacity-related term is also important in describing the asymmetry of the osmotic compressibility as it was observed in studies of the diameters of the coexistence curves.

Critical anomalies of alkaline fading of phenolphthalein in the critical solution of 2-butoxyethanol + water.

We have used three-wavelength UV-spectrophotometry to study the reaction of the alkaline fading of phenolphthalein in the critical solution of 2-butoxyethanol + water. It was found that when the temperature was far away from the critical point, the values of the natural logarithm of the rate constant k and the natural logarithm of the chemical equilibrium K determined in our experiments had good linear relationships with the reciprocal of temperature, which served as the backgrounds and were used for correcting k and K in the critical region. The critical slowing down of the reaction and the critical anomaly of the chemical equilibrium were detected near the critical point. The value of the critical exponent characterizing the slowing down effect of the reaction rate was obtained to be 0.156, which was close to the value 0.11 associated with the heat capacity divergence and agreed with the theoretical prediction. The experimental result also confirmed the theoretical prediction of 0.11 for the critical exponent characterizing the weak divergence of the singularity of the chemical equilibrium.

Absolute quantification of single-base m6A methylation in the mammalian transcriptome using GLORI.

N6-methyladenosine (m6A) is the most abundant RNA modification in mammalian cells and the best-studied epitranscriptomic mark. Despite the development of various tools to map m6A, a transcriptome-wide method that enables absolute quantification of m6A at single-base resolution is lacking. Here we use glyoxal and nitrite-mediated deamination of unmethylated adenosines (GLORI) to develop an absolute m6A quantification method that is conceptually similar to bisulfite-sequencing-based quantification of DNA 5-methylcytosine. We apply GLORI to quantify the m6A methylomes of mouse and human cells and reveal clustered m6A modifications with differential distribution and stoichiometry. In addition, we characterize m6A dynamics under stress and examine the quantitative landscape of m6A modification in gene expression regulation. GLORI is an unbiased, convenient method for the absolute quantification of the m6A methylome.

Spectrometric study of AOT-hydrolysis reaction in water/AOT/isooctane microemulsions using phenolphthalein as a chemical probe.

The kinetics of the alkaline hydrolysis of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in water/AOT/isooctane microemulsions has been studied by monitoring the absorbance change of the phenolphthalein in the system with time. The apparent first-order rate constant k(obs) has been obtained and found to be dependent on both the molar ratio of water to AOT ω and the temperature. The dependences of k(obs) on ω have been analyzed by a pseudophase model which gives the true rate constants k(i) of the AOT-hydrolysis reaction on the interface and the partition coefficients K(wi) for the distribution of OH(-) between aqueous and interface pseudophases at various temperatures; the latter is almost independent of the temperature and ω. The temperature dependences of the reaction rate constants k(obs) and k(i) have been analyzed to obtain enthalpy ΔH(≠), entropy ΔS(≠), and energy E(a) of activation, which indicate that the distribution of OH(-) between aqueous and interface pseudophases increases ΔS(≠) but makes no contribution to E(a) and ΔH(≠). The influence of the overall concentration of AOT in the system on the rate constant has been examined and found to be negligible. It contradicts with what was reported by García-Río et al. (1) but confirms that the first-order reaction of the AOT-hydrolysis takes place on the surfactant interface. The study of the influence of AOT-hydrolysis on the kinetics of the alkaline fading of crystal violet or phenolphthalein in the water/AOT/isooctane microemulsions suggests that corrections for the AOT-hydrolysis in these reactions are required.