The parameters reported herein will help predicting RNA duplex stability in different crowded environments, which will lead to an improved understanding of the stability-function relationship for RNAs in various cellular organelles with different molecular environments.RNA performs various spatiotemporal functions in living cells. As the solution environments significantly affect the stability of RNA duplexes, a stability prediction of the RNA duplexes in diverse crowded conditions is required to understand and modulate gene expression in heterogeneously crowded intracellular conditions. Herein, we determined the nearest-neighbor (NN) parameters for RNA duplex formation when subjected to crowding conditions with an ionic concentration relevant to that found in cells. Determination of the individual contributions of excluded volume effect and water activity to each of the NN parameters in crowded environments enabled prediction of the thermodynamic parameters and their melting temperatures for plenty of tested RNA duplex formation   RNA is one of the most important biomolecules present in cells and is associated with various biological processes, such as protein synthesis, biocatalysis, and enzymatic reactions ,2. RNA cG°) for the duplex formation comprises the sum of the ΔG° values of adjacent NN base pairs and helix initiation (H°) and entropy (ΔS°), can also be predicted from the base sequence. Based on this model, parameters for predicting the thermodynamics of RNA, DNA and RNA/DNA hybrid duplex formations were developed  are predominantly used  model, which assumes that the free energy change . Based on these data, we determined the NN parameters of 10 possible propagating Watson–Crick NN base pairs and helix initiation factor for RNA duplexes, which predicted stability of RNA duplexes with high accuracy even in a solution containing exact intracellular cation composition. Furthermore, we expanded these NN parameters available under different cosolutes at varying concentrations by determining the contributions of the excluded volume effect and water activity under the cosolute-containing solutions on duplex stability, which permitted to predict stabilities of different RNA duplexes in diverse in vitro and intracellular crowding conditions with significant accuracy. The universal NN parameters applicable to different crowding conditions will be not only useful in RNA structure determination software when attempting to improve our understanding of RNA structures in vivo, but also in designing RNA-based therapeutics.In this study, we analyzed the thermodynamics of formation of different RNA duplexes with varying lengths and base compositions in a physiologically relevant solution containing 100 mM NaCl and 40 wt% PEG with an average molecular weight of 200 g mol2HPO4), Dipotassium hydrogen phosphate (K2HPO4), sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl2) and calcium chloride (CaCl2) were purchased from Wako Pure Chemical Industries (Japan); disodium ethylenediaminetetraacetate (Na2EDTA) and dipotassium ethylenediaminetetraacetate (K2EDTA) were purchased from Dojindo Molecular Technologies (Japan) and all these chemicals were used as received.All the synthetic RNA oligonucleotides used in this work, listed in Table S1 (in Supplementary Data), were purchased from Japan Bio Services Co. and purified by high-performance liquid chromatography (HPLC). Dialysis could be done to set the exact ionic concentrations of the studied solutions, however, we did not perform dialysis, except otherwise mentioned. This is due to the fact that we used quite short length RNAs with low concentrations for this study. Thus, even if there were some residual cations on RNAs after synthesis and purification, the effect on the stability should be negligible. RNA samples were prepared in Milli-Q water and stocked at -20°C until use. The concentrations of the single-stranded oligonucleotides were determined by measuring the absorbance at 260 nm at 90°C using the extinction coefficients . Polyeth2HPO4, 1 mM Na2EDTA and 100 mM NaCl, in the presence of cosolutes with specific weight percentages. We adjusted the pH of the buffer to 7.0 after adding the cosolutes to maintain the pH of the buffer solution. For melting experiments, 10–12 fresh solutions of oligonucleotides were prepared by varying the concentrations over a 50–150-fold range. The RNA solutions were kept at 90°C for 5 min, followed by the decrease of temperature from 90°C to 0°C at a rate of 1°C min−1 to anneal the duplexes. Thereafter, the samples were heated from 0°C to 90°C at a rate of 0.5°C min−1 to melt the duplex after keeping them at 0°C for 5 min. Water condensation on the cuvette exterior at low temperature was avoided by flushing with a constant stream of dry N2 gas.Absorption spectra were measured on a Shimadzu 1800 spectrophotometer with a thermoprogrammer. All the experiments were conducted in a buffer containing 10 mM NaH°, ΔS° and ΔG°37) for RNA duplexes were determined from the mT−1 versus ln (Ct/s) plots as described in our earlier studies  or non-self-complementary strands (s = 4). Following the standard practice for calculation of the thermodynamic parameters, we assumed the difference in heat capacities (ΔCp) of the two states (single-strand and duplex) to be zero (mT) values for most of the studied sequences in the crowding condition were not far from 37°C, zero ΔCp approximation should be acceptable for ΔG°37 calculation due to minimal extrapolations. Generally, ΔG°37 is relatively insensitive to ΔCp due to enthalpy–entropy compensation  by applying linear least square fitting to the measured values of the thermodynamic parameters of 45 RNA duplexes in 40 wt% PEG200 with 100 mM NaCl. The parameters for symmetry correction were set to the same value as that in the absence of cosolutes, since it does not depend on the solution condition, but on the complementarity of the sequences. ΔS° values were obtained from the determined respective values of ΔG°37 and ΔH° using Equation NN parameters were calculated using our original software written in Python based on the linear least square fitting algorithm as described in our earlier studies ,31. Brie2 gas to avoid water condensation on the cuvette exterior. The CD spectra were measured from 200 to 340 nm in 1.0 mm path-length cuvettes with a scan rate of 50 nm min−1. The concentration of the samples was 20 μM in a buffer containing 100 mM NaCl, 10 mM Na2HPO4 (pH 7.0) and 1 mM Na2EDTA with or without 40 wt% PEG200.CD spectra were obtained on a JASCO J-1500 spectropolarimeter equipped with a temperature controller. The experimental temperature was 4°C. The cuvette-holding chamber was flushed with a constant stream of dry Naw) was calculated from the measured osmomolality (mmol kg−1) using the following equation (The water activities were determined by vapor phase osmometry (a Wescor pressure osmometer) or freezing point depression osmometry (a Knauer osmometer). The water activity , respectively. The relationship between water potential and osmomolality is given by the following equation (where Ψ and equation The dielectric constants were measured by using fluorescent probe ANS. The fluorescence maximum of ANS showed a hypsochromic shift in the media of low dielectricity. The spectra were recorded in fluorescence spectrophotometer (Jasco F-6500) exciting at 370 nm. The dielectric constants were calculated using a standard curve of organic solvents with known values.We selected RNA sequences ranging between 6- and 12-mers, ensuring sufficient stability and two-state transitions, since short sequences are too unstable to analyze, and longer sequences may show non-two-state transitions by forming intramolecular structures. The chosen sequences had different combinations of NN frequencies, covering all 10 NN base pairs for RNA duplex formation, without any bias to particular pairs (Table S1). We selected 25 non-self-complementary (NS1–NS16) and 20 self-complementary (S1–S15) RNA duplexes, as shown in Table S1. Among the duplexes investigated, nine pairs of non-self-complementary sequences (NS7–NS15) and five pairs of self-complementary sequences (S9–S12 and S15) had identical NN base sets (marked as ‘a’ and ‘b’ in Table S1). CD spectral data of representative RNA duplexes indicated that these sequences adopted the A-form duplex both in the absence or presence of PEG200 . The sliH°, ΔS° and ΔG°37) of RNA duplex formation for the studied sequences under physiological crowding condition containing 40 wt% PEG200 with 100 mM NaCl were determined from the UV melting experiment, as depicted in Figure + concentration of the experimental solution was 122 mM considering the Na+ derived from the buffer components (10 mM Na2HPO4 and 1 mM Na2EDTA) in addition to 100 mM NaCl. Consistent with previous reports, the stability of all the RNA duplexes decreased under crowding condition due to changes in the solution properties such as water activity and the dielectric constant of the medium in the presence of a cosolute (Tm−1 versus ln (Ct/4) plots under physiological crowding condition , the average differences in the percentage of measured ΔH°, TΔS° and ΔG°37 were 5.2%, 5.8% and 4.0%, respectively. Since the NN model assumes the same stability for sequences with identical NN pairs  and the penalties for helix initiation and terminal A–U pairs were calculated using a linear least-square fitting algorithm from the thermodynamic parameters of 45 RNA sequences (Table S2)  was: r(AU/UA) ≈ r(AA/UU) < r(UA/AU) < r(CU/GA) ≈ r(GU/CA) < r(CA/GU) ≈ r(CG/GC) ≈ r(GA/CU) < r(GG/CC) < r(GC/CG). The quantitative effect of molecular crowding on the individual NN pairs was estimated by subtracting parameters in the absence of a cosolute of the same cation concentration (The parameters for 10 possible Watson–Crick NN pairs (Δable S2) . Table 1ntration . Each bantration . For fou−1, respectively, in the 40 wt% PEG200 solution (Table S4). This suggests that the formation of very short RNA duplexes under conditions of molecular crowding may be hindered due to the large unfavorable energy required for forming the first base pair in the duplex, while several NN pairs may be needed to compensate for this energy. The hydration around the terminal of a duplex is different from that of the stem as water molecules around the terminals are more accessible, resulting in greater disruption by the crowders. Therefore, the relative effect of crowders on NN pairs depends on the hydration around the particular NN pair.The effect of crowding was significant for helix initiation and terminal A–U pairs. Helix initiation and terminal A–U pairs were destabilized by 1.41 and 0.40 kcal molH°, ΔS°, ΔG°37 and Tm between the predicted and measured values among the 45 studied RNA duplexes were 5.0%, 5.5%, 3.5% and 1.1°C, respectively (Table S5), which were well within the error limits reported in previous studies  into the individual contributions of the bulk interactions  and environmental factors  comprising of the energetic contribution of the cation  and crowder . Therefore, each NN pair (ΔG°NN) under crowding conditions can be expressed as follows:The molecular environments in cell vary spatiotemporally with the cell cycle, leading to different crowding densities . MoreoveG°NN is equal to ΔG°NN, bulk in the absence of contributions from ΔG°NN, cation and ΔG°NN, crowder. In the absence of a crowder, ΔG°NN showed exponential decay with increasing concentration of Na+ .The bulk interactions consist of hydrogen bonding in base pairs, stacking interactions between neighboring base pairs, and conformational entropic penalty to bring two single strands into a helical structure . Accordin of Na+ . Howevertrations  37). Th. ThG°NN ntration . Thus, Δntration . The ΔG°G°NN, crowder, individual contributions from the excluded volume effect  and water activity  should be quantified since those are the dominant physical and chemical factors, respectively, for the duplex stability in cosolute solutions. was determined using the following equation: , ρ is the density of water in kg L−1, ΔV is the change in the excluded volume between the nucleic acid and the cosolute upon duplex formation in L mol−1, and C is the molal concentration of the cosolute in mol kg−1 , and k is a geometrical factor derived from the length and radius of the RNA in Å (Table S6). The factor 10−27 is used for the conversation of Å3 to L. Using the parameters listed in Table S6, we calculated V/N for different lengths of RNA in PEG using Equation V/N in Table S6, ΔV/N and ΔG°37, ev, dup/C•N were calculated using Equations G°37, ev, dup/C•N decreased as the length of the RNA increased  suggested that Equation G°37, ev, dup for any given length of RNA duplex and cosolute concentration. Assuming that all NN pairs have the same volume effect, the contribution of the excluded volume effect for each NN pair and initiation factor  was determined by the relation below  for the test sequence in different cosolute solutions was calculated by subtracting the stability in the absence of cosolute  and ΔG°37, ev, dup from the measured ΔG°37 (Table S8). For example, in 10 wt% EG solution at 100 mM NaCl, ΔG°37, wa, dup for GAUUACGCCUG was calculated to be 0.95 kcal mol−1 by as follows: ΔG°37, wa, dup = ΔG°37 − ΔG°37, no cosolute - ΔG°37, ev, dup, where ΔG°37, ΔG°37, no cosolute and ΔG°37, ev, dup were −13.6, −14.1 and −0.45 kcal mol−1, respectively (Table S8). The plot of ΔG°37, wa, dup against the changes in water activity, Δaw (Δaw =ano cosolute − acosolute) revealed two different linear correlations depending on the chemical structures of the cosolutes  is also linearly correlated with the water activity of the solution by the following relation:To explore the relationship between the RNA duplex stability and the water activity of the solution, we measured the stability of a test RNA sequence in solutions with different water activities as obtained by different cosolutes at varying concentrations (Table S8). We selected GAUUACGCCUG as a test sequence because it contained all 10 NN base pairs with a single occurrence, which eliminated bias toward any particular NN pair. The contribution of water activity to the duplex , cation  and excluded volume effect  from the ΔG°37, NN parameters of Table G°37, NN, wa for 40 wt% PEG200, as shown in Table mcs values for PEG (mPEG) were calculated from the ΔG°37, NN, wa [40 wt% PEG200] values using Equation mcs values of the other cosolutes were calculated as follows:where °37, NN, 22 mM Na+SCS and SPEG are the slopes for the cosolute of interest and PEG, respectively, in the ΔG°wa, dup vs. Δaw plot  in different cosolute solutions are available in the literature or can be measured by osmometry, as mentioned in the Supplementary data. Therefore, the stability of RNA duplexes in different crowded environments can be predicted from their base sequences, using the universal parameters listed in Table where t Figure . ΔG°37, cs Table  and Δaw.+-containing solutions, the intracellular environment contained K+ as the major monovalent cation . This could be as a result of the different binding modes of the two cations with the RNA , favoring electrostatic interaction between negatively charged phosphates and Na+. However, in the absence of cosolute and in 40 wt% EG, where dielectricity did not decrease much, RNA duplexes showed only a marginal increase in their stability in the presence of Na+ compared with K+ with an average differences of 3.1% and 4.2%, respectively (Tables S11 and S12). We investigated the quantitative relationship of RNA stability between K+ and Na+ in 40 wt% PEG200 to obtain duplex stability in the presence of K+ from their values in the Na+ solution. We observed a good linear relationship between the measured stability in K+ and the predicted stability in the same concentration of Na+ solution using our derived parameters  is zero. This excess energy contribution is mainly affected by structural factors, such as duplex terminals. Positive intercept suggested that, under crowding conditions, K+ destabilizes the duplex terminals on average by 0.45 kcal mol−1 compared to Na+. Using Equation G°37 of the 16 duplexes in the K+ solution showed an average difference of only 4.3% from the measured values (Table S13), suggesting that, without calculating the NN parameters in the KCl condition, our derived parameters in 100 mM NaCl solution can provide an accurate estimation of RNA stability in 100 mM KCl following a simple linear relation.The slope of the equation is defined as the ratio of the change in Δulations . The intG°37 for a sequence in a particular cosolute solution using generalized parameters is illustrated in Figure G°37 values for the 52 sequences listed in Table S14 was 7.3%. Therefore, these results indicate that the generalized parameters can predict RNA duplex stability under different NaCl conditions and in various cosolutes, differing largely in their molecular weights, with significant accuracy.We verified the applicability of the generalized parameters for different crowding conditions using a large number of RNA sequences with different NN compositions and lengths. We used the parameters in Table + and K+, and small amounts of divalent cations such as Mg2+, the approximation of ΔG°NN, cation is needed because the database of ΔG°NN, cation values was available for a single component of the cation. To verify the applicability of the derived parameters under intracellular cation conditions, we measured thermodynamic parameters for the same 16 RNA duplexes in a buffer solution containing the exact intracellular cation composition  (Tm of the duplexes measured in 122 mM Na+ with 40 wt% PEG200 (Table S15), indicating that the combined effect of monovalent and divalent cations on the duplex stability at the intracellular concentration is similar to that of the Na+ condition used for determining the NN parameters. When we plotted the measured ΔG°37 in the 40 wt% PEG200 with the intracellular cation concentration against the corresponding predicted values in the Na+ solution with 40 wt% PEG200 , we obtained a straight line with the following equation.For the established parameters to predict RNA duplex stabilities in cells, which contain monovalent cations such as NamM Ca2+) ,50 under+. However, contrary to Equation −1, suggesting that duplex terminals were stabilized by intracellular cation concentration as Mg2+ can bind to the N7 atom of the exposed purine bases at terminals , validating the applicability of the derived NN parameters for intracellular cation conditions. Therefore, molecular crowding conditions with 122 mM Na+ should be ideal as a model solution for studying physiological crowding in cells for physicochemical measurements.Equation erminals . The comG°25 (difference in ΔG°25 between inside and outside the nucleolus) for two RNA duplexes, (ACUG)3 and (ACUG)6 were measured to be 3.0 and 3.3 kcal mol−1 at 25°C, respectively. Since, these destabilizations were mainly due to the crowding condition inside the nucleolus, it can be considered as ΔG°[crowder] at 25°C. We used our generalized ΔG° parameters adjusted for 25°C using ΔH° and ΔS° values (see Table S17) to determine the cosolute condition that imposes similar destabilizations, as reported for the two duplexes. We found that using our generalized parameters the measured destabilizations were matching with the predicted ΔG°25, [crowder] in 40∼50 wt% PEG200 solution (see Table S18), which is the same condition predicted using our DNA NN parameters for mimicking nucleolus  hairpin sequence located in 5′-UTR of the agsA gene  from their stability in the absence of cosolute  and change in the water activity of cosolute solution (Δaw) as follows:Moreover, we demonstrated the prediction of hairpin RNA (Im-4U), mutated from gsA gene . In Im-4gsA gene . For G–UgsA gene . For loogsA gene , we form−1 . From these parameters and relationship Equation , the sta−1 . The preG°37 of Im-4U in the nucleus and cytosol of live HeLa cells were reported to be –1.7 and –2.6 kcal mol−1, respectively  and did not match the reported ΔG°37 inside HeLa cells. Thus, we used less destabilizing cosolutes  to estimate the intracellular crowding in vitro. We identified that the ΔG°37 of Im-4U in the nucleus and cytosol of HeLa cells were successfully predicted to be –1.9 and –2.7 kcal mol−1, respectively, using our parameters in 30 wt% and 40 wt% 1,3-PDO conditions at 100 mM NaCl (Table S21). These solution conditions were different from that of the nucleolus, as mentioned above, indicating that our prediction is versatile in different localized cellular regions. In contrast, it should be noted that the experimental ΔG°37 exhibited a wide distribution of about 2.0 kcal mol−1 in the measured values in the nucleus and cytosol  ΔG°37, NN, bulk from Table G°37, NN, Na+ from Table G°37,NN, ev from Table G°37,NN, wa determined from the mCS values in Table G°37,NN, ev and ΔG°37,NN, wa terms, specific NN parameters for the nucleolus condition using 40 or 50 wt% PEG200 in 100 mM NaCl solution and the cytosol condition using 40 wt% 1,3 PDO with 100 mM NaCl can be obtained. The users can freely arrange the parameters for predictions in their specific conditions containing different concentrations of Na+ and crowders using these table and equations (as demonstrated in Figure We developed prediction parameters of in vitro and in cells. Our approach should be beneficial for not only investigating biological processes but also for designing RNA structures for RNA interference, antisense oligonucleotide therapy, and mRNA vaccines, which work efficiently in specific environments of different cells. Moreover, our method can provide the information of in-cell secondary structures which is currently unknown to develop RNA targeting drugs for RNA viruses including SARS-CoV-2.A prediction of the thermodynamic stability of RNA duplexes under diverse crowding conditions is a basis to determine RNA folded structures in spatiotemporal area in cells. Further collection of parameters of various structural motifs like bulges, mismatches, and dangling ends will enable to predict large RNA structures All relevant data are included in the paper and/or its supplementary data.gkad020_Supplemental_FileClick here for additional data file.


Paramyxoviridae family and closely related to the Mojianghenipavirus, has raised concerns among public health authorities worldwide. The coronavirus 2019 (COVID-19) pandemic, which started in China in 2019, is still ongoing, despite joint global efforts [The newly-emerged Langya virus (LayV), classified as a henipavirus in the Most disease outbreaks during the past two decades have been of zoonotic origin . AlthougLayV has emerged as a threat following the Hendra and Nipah viruses, taking its name after a town called Langya in China. From its emergence in 2018, 35 cases of LayV infection have been reported, all in eastern China. To date, no deaths have been reported. Despite its low global prevalence, this novel virus could lead to a global public health crisis, especially due to the potential high mortality from earlier species of the henipavirus genus, which is why it cannot be underestimated ,8. CurreLangya virus is an enveloped virus containing single-stranded ribonucleic acid (RNA) with 18 402 RNA nucleotides. Its genomic structure consists of six structural proteins – nucleocapsid, phosphoprotein, matrix protein, surface glycoprotein, fusion protein, and large viral RNA-dependent RNA polymerase. These six structural proteins determine its closest genetic linkage with other henipaviruses like Mojiang henipavirus [As previously mentioned, the virus primarily transmits between animals, most likely among shrews. The LayV RNA was isolated from more than 25% of almost 260 shrews studied, leading researchers to conclude that the species may possibly serve as a direct or indirect vector for human transmission. The LayV RNA has also been isolated from other animals, including dogs and goats . Among 3Farmers and manufacturing workers are the populations most vulnerable to LayV ,11, as r


Here, we introduce a genetically encoded CAG-repeat RNA tag to reprogram cellular condensate formation and recruit various non-phase-transition RNAs for cellular modulation. With the help of fluorogenic RNA aptamers, we have systematically studied the formation dynamics, spatial distributions, sizes and densities of these cellular RNA condensates. The cis- and trans-regulation functions of these CAG-repeat tags in cellular RNA localization, life time, RNA–protein interactions and gene expression have also been investigated. Considering the importance of RNA condensation in health and disease, we expect that these genetically encodable modular and self-assembled tags can be widely used for chemical biology and synthetic biology studies.Living systems contain various membraneless organelles that segregate proteins and RNAs via liquid–liquid phase separation. Inspired by nature, many protein-based synthetic compartments have been engineered   The functions of cellular RNAs are highly related to their subcellular localizations and local environment. One ubiquitous approach to control RNA localization is via macromolecular condensation, i.e. the formation of membraneless subcellular compartments. RNA compartmentalization is prevalent and plays critical roles in processes such as transcription, splicing, RNA degradation, heterochromatin formation and stress response . The preCellular RNA localization, compartmentalization and trafficking have been largely regulated by the formation of ribonucleoprotein (RNP) complexes via RNA–protein interactions ,6. Recencis-acting RNA element within the target RNA transcript or a trans-acting effector functioning through specific hybridization with target RNAs. Both cis- and trans-mechanisms can be potentially applied to develop general platforms to induce the phase separation of various target RNAs, in vitro and in living systems.In this study, short CAG trinucleotide repeats are engineered into genetically encodable self-regulated tags to recruit and condense different cellular target RNAs. CAG repeats are used here because these trinucleotides can readily form condensates, without the involvement of proteins ,10,13,14To image these RNA condensates, especially inside living cells, we fuse the CAG-repeat-tagged RNA strands with a fluorogenic RNA aptamer, named Broccoli. Broccoli is an RNA strand that can selectively bind and activate the fluorescence signal of small molecule dyes, such as DFHBI-1T . The dynThe reagents, apparatus and RNA sequences used in this study are listed in the Supplementary Data.Escherichia coli BL21 Star™ (DE3) cells (Thermo Fisher Scientific) and selected based on ampicillin resistance.Different lengths of the CAG repeats were first cloned into a pAV-U6 + 27-F30-2×dBroccoli vector. In more detail, the vector was first digested with XbaI and SacII restriction enzymes (NEB). After purification via a 1% agarose gel, the digested vector was ligated together with a similarly digested CAG repeat insert using T4 DNA ligase (NEB). The ligated product was then transformed into To construct CAG repeat-expressing pET-28c vectors for bacterial imaging, a pET-28c-F30-2×dBroccoli vector was digested with BgIII and XhoI restriction enzymes (NEB), and then ligated together with a similarly digested CAG repeat insert using T4 DNA ligase. The CAG repeat inserts were synthesized by polymerase chain reaction (PCR) using the above-prepared pAV-U6 + 27-F30-2×dBroccoli-CAG repeat vectors as the template, primers containing the T7 terminator, and BgIII and XhoI restriction sites. After T4 DNA ligation, the product was transformed into BL21 Star™ (DE3) cells and selected based on kanamycin resistance.lacY and lacZ fragments were each cloned into pET-28c-F30-2×dBroccoli-CAG repeat vectors after digestion via BsaI and EcoRI restriction enzymes (NEB). The lacY and lacZ fragments were synthesized by PCR using the pYFP-lacY-1 and pSFV3-lacZ plasmids as the templates, and primers that introduce BsaI and EcoRI restriction sites. After T4 DNA ligation, the product was transformed into BL21 Star™ (DE3) cells and selected based on kanamycin resistance. All these above-prepared plasmids were isolated using a GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific) and confirmed by Sanger sequencing (Eurofins Genomics).The OxyS, in vitro experiments, all the RNAs were transcribed using a HiScribe™ T7 high-yield RNA synthesis kit (NEB) and purified with G-25 columns. Template DNAs were prepared by PCR amplification from the above-mentioned pAV-U6 + 27- or pET-28c-based F30-2×dBroccoli-CAG repeat vectors. The in vitro formation of RNA condensates was prepared by first mixing 4 μM RNAs in buffer containing 10 mM Tris–HCl at pH 7.5, 100 mM KCl and 20 mM MgCl2. The mixture was then heated up to 95°C for 3 min and cooled down to 37°C at a rate of 2°C/min in a thermocycler. Afterwards, 80 μM DFHBI-1T was added and incubated for 15 min at 37°C before imaging using a Yokogawa spinning disk confocal on a Nikon Eclipse-TI inverted microscope. Images were collected with an excitation wavelength at 488 nm using a ×100/1.45 NA oil immersion objective. The partition ratio of each RNA condensate was calculated based on the ratio of average fluorescence intensity within the condensate versus that in the solution region free of condensates. For the in vitro kinetic measurements, a solution containing 10 mM Tris–HCl at pH 7.5, 100 mM KCl, 20 mM MgCl2, 4 μM RNA and 80 μM DFHBI-1T was first heated up at 95°C for 3 min and then rapidly cooled down in ice for 30 s immediately before starting to collect images.For Pseudomonas aeruginosa cells that express the corresponding RNAs were first grown in LB medium at 37°C until the optical density at 600 nm (OD600) reaches 0.4, and then 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) was added for a 2 h induction. After the IPTG induction, the cells were adhered to a poly-l-lysine-pre-treated glass-bottom 8-well imaging plate  in Dulbecco’s phosphate-buffered saline (DPBS) buffer for 45 min. Then the buffer was switched to fresh DPBS containing 200 μM DFHBI-1T and/or 1 μM HBC620 for a 30 min incubation at 25°C before imaging. All the confocal fluorescence images were collected with NiS-Elements AR software using a Yokogawa spinning disk confocal on a Nikon Eclipse-TI inverted microscope. Broccoli fluorescence signals were excited with a 488 nm laser, Pepper fluorescence signals were excited with a 561 nm laser and the near-infrared fluorescent protein signal was collected under 640 nm laser irradiation. A ×100/1.45 NA oil immersion objective was used for collecting these cellular images. Structured illumination microscopy (SIM) super-resolution imaging was performed on a Nikon A1R-SIMe microscope equipped with a Hamamatsu sCMOS camera and a ×100 oil immersion objective, under 488 nm laser irradiation.RNA imaging in living bacterial cells was performed according to a previously established protocol . BrieflyThe FRAP measurements were performed on an A1 spectral confocal microscope with a ×100 oil immersion objective on a Nikon Eclipse-TI inverted system with an A1 stimulation module to ensure bleaching of a targeted area. Photobleaching was carried out with a 488 nm laser for 1.0 s on a region of ∼1 μm diameter. The fluorescence recovery after bleaching was then imaged every 5 s for a total of 3–5 min.GA3) module. For in vitro fluorescence images, a fluorescence intensity ‘threshold’ FB + 3SD (background plus 3-fold of standard deviations on the 488 nm channel) and a diameter ‘threshold’ of 0.5 μm was set to automatically detect each condensate. For each identified condensate, the ‘mean object intensity’ and ‘object area’ actions were applied to measure their corresponding mean fluorescence intensity and the area. The number of identified objects in one image (imaging view 4430 μm2) equals the measured density of condensates. An inverted threshold was also set for measuring mean background intensities for the calculation of partition ratios.Image analysis was performed using NiS-Elements AR Analysis software. The sets of actions performed on imaging channels were built as analysis recipes in the General Analysis 3 (B + 3SD (whole well background plus 3-fold of standard deviations on the 488 nm channel) was set to automatically detect each individual E. coli cell. The averaged whole-cell fluorescence was measured via the ‘mean object intensity’ action. To detect intracellular foci, the ‘bright spots’ action was applied based on a fluorescence intensity ‘threshold’ FB + 3SD (cellular background plus 3-fold of standard deviations on the 488 nm channel) and a diameter ‘threshold’ of 0.5 μm. ‘Contrast’ and ‘grow’ were also set to enable the proper detection of foci. Meanwhile, the ‘child ID’ action was applied to set the detected cells as ‘parent’ and the detected foci as ‘child’ and chose ‘child is inside parent’ condition. The count of foci in each detected cell was then obtained using the ‘group records’ and ‘aggregate rows’ actions. The ‘subtract’ action was used to subtract the detected foci region from the detected cells region, i.e. the cellular background region. Then, the ‘mean object intensity’ action was applied on both the foci region and the cellular background region to measure their corresponding mean fluorescence intensities and the partition ratio of each cellular condensate.For bacterial fluorescence images, after applying a ‘smooth’ action, a fluorescence intensity ‘threshold’ Ft-test was used to determine the statistical significance.To further avoid inappropriate detection of cells and foci, only rod-shaped singly detected cells with properly detected foci were picked and also manually verified. The results shown in this study were generated by at least 90 above-verified cells from at least three representative images unless mentioned otherwise. At least two replicated experiments were performed for all these measurements. All the data analysis and fitting were performed using the ImageJ/FIJI and GraphPad Prism 9.2.0 software. Two-tailed Student's 2 and 80 μM DFHBI-1T, the 20R, 31R and 47R samples exhibited obvious RNA condensation . Co. Co17). n Figure , with a n Figure . In cont2 imaging area) than that of 20R, 31R and 47R (>15 counts per same area)  long scrambled sequences. The 0×, 20×, 31× or 47× CAG repeats were conjugated at the 3′ end of these strands, respectively. The multiple of 4× CAG repeats was not chosen because condensation occurs with low efficiency. Without attaching CAG repeats, scrambled RNAs  could not form condensates in a solution containing 4 μM RNA, 20 mM MgClIn contrast, the 20× CAG tag can induce compartmentalization when short RNA sequences (i.e. 100 and 200 nt) were attached, while obvious condensates were shown in all the solutions containing 31× CAG- or 47× CAG-tagged RNA strands. These results indicated that CAG repeats can still induce phase separation even after tagging with long non-condensation RNA strands.We further quantified the correlations between the length of CAG repeats and the partition ratio and size of condensates. As shown in lac operon mRNAs, lacY (1254 nt) and lacZ (3044 nt). After tagging with F30-2d×Broccoli and 20×, 31× or 47× CAG repeats, OxyS can easily form obvious condensates, with size and partition ratio comparable with those of 0.1k scrambled RNAs  and two s, lacY 154 nt ands Figure . Similard Figure . All theCAG/Ntotal  E. coli cells with pET-28c vectors that express 4R, 20R, 31R or 47R sequences. Compared with the control cells encoding only F30-2d×Broccoli (0R) or 70AC, the formation of cellular condensates can be clearly visualized in CAG-repeat-containing strains  contain one or two condensates (76–85%), mainly localized at the cell poles. Meanwhile, it is worth mentioning that minimal cytotoxicity was observed in these CAG repeat-expressing li cells . Indeed,in vitro data  cells, bright fluorescent foci can be observed only in CAG-repeat-expressing cells  that encode a near-infrared fluorescent protein (NirFP). Compared with Nir-0R cells, ∼20% lower NirFP signals were observed in Nir-47R cells , while the majority of translated NirFP proteins were observed throughout the cells, except the poles. We further plotted the cellular NirFP signals as a function of the number of condensates in each Nir-47R cell. A reduced NirFP signal was shown in cells that contain more condensates  cells with vectors expressing 0× CAG- or 47× CAG-tagged 705 nt long mRNA  that were tagged with a complementary sequence that hybridizes with either an OxyS or lacY target RNA. A 23 nt and a 26 nt long non-structural region in OxyS and lacY was respectively designed as the targeting domain, whose secondary structures were pre-evaluated via Mfold and NUPACK software  cells. Similar to that shown in Broccoli-tagged CAG-repeat-expressing cells  together with an F30-2d×Broccoli-conjugated 47× CAG-repeat strand (47R) or that contains a 25 nt long complementary sequence 47R-cN. Despite some spectral overlaps, the NirFP fluorescence and Pepper/HBC620 signals can still be imaged simultaneously with negligible cross-talk  and gene expression patterns (in the case of NirFP and OxyS), can also be regulated by these CAG-repeat tags.The importance of RNA condensation in studying cellular functions as well as disease diagnosis and treatment has been increasingly recognized ,33. ProgWe expect that these functional CAG-repeat tags can be broadly applied to reprogram living cells with defined compartmentalization and structures. This work can also inspire the potential engineering of various types of genetically encodable RNA nanodevices, which can be based on similar nature-inspired RNA repeat tags. These tags will be used to regulate different endogenous RNA targets and may also be applied to orthogonally recruit different RNA and protein molecules into specific RNA condensates. Powerful nanodevices may also possibly be designed to be controllable by different small-molecule or light triggers.gkad621_Supplemental_FileClick here for additional data file.


UBE2S was positively correlated with the lymphatic metastasis status, high tumor stage, histological grade, and poor prognosis of BCa patients. Functional assays showed that UBE2S promoted BCa cell migration and invasion in vitro, as well as lymphatic metastasis in vivo. Mechanistically, UBE2S interacted with tripartite motif containing 21 (TRIM21) and jointly induced the ubiquitination of lipoma preferred partner (LPP) via K11-linked polyubiquitination but not K48- or K63-linked polyubiquitination. Moreover, LPP silencing rescued the anti-metastatic phenotypes and inhibited the epithelial-mesenchymal transition of BCa cells after UBE2S knockdown. Finally, targeting UBE2S with cephalomannine distinctly inhibited the progression of BCa in cell lines and human BCa-derived organoids in vitro, as well as in a lymphatic metastasis model in vivo, without significant toxicity. In conclusion, our study reveals that UBE2S, by interacting with TRIM21, degrades LPP through K11-linked ubiquitination to promote the lymphatic metastasis of BCa, suggesting that UBE2S represents a potent and promising therapeutic target for metastatic BCa.Lymphatic metastasis is the most common pattern of bladder cancer (BCa) metastasis and has an extremely poor prognosis. Emerging evidence shows that ubiquitination plays crucial roles in various processes of tumors, including tumorigenesis and progression. However, the molecular mechanisms underlying the roles of ubiquitination in the lymphatic metastasis of BCa are largely unknown. In the present study, through bioinformatics analysis and validation in tissue samples, we found that the ubiquitin-conjugating E2 enzyme  As one of the most common malignancies of the genitourinary system, bladder cancer (BCa) had 573,000 newly diagnosed cases and 213,000 deaths worldwide reported in 2020 . In addiPosttranslational modification (PTM) is an important regulatory process for cellular proteins and is extensively involved in physiological and pathological functions, including apoptosis, innate immunity and cancer –14. As oUBE2S) has been reported to be upregulated in various human cancers and correlates with a poor prognosis. In hepatocellular carcinoma, Zhang et al. reported that UBE2S interacted with intranuclear TRIM28 to accelerate the cell cycle by inducing proteasomal degradation of p27 to support hepatocellular carcinoma development [UBE2S was responsible for the degradation of p16 and stabilization of β-catenin via K11-linked polyubiquitination to promote bone metastasis [UBE2S in lymphatic metastasis of BCa remain to be clarified.Ubiquitin-conjugating enzyme E2S  to form K11-linked polyubiquitination chains and mediated lipoma preferred partner (LPP) degradation, which finally promoted the epithelial-mesenchymal transition (EMT) and enhanced the lymphatic metastasis of BCa. Targeting UBE2S with cephalomannine might be a potential therapy to effectively reduce the lymphatic metastasis of BCa.In this study, we revealed that Approval for the use of human tissues was obtained from the ethics committee of Sun Yat-sen University. Five paired fresh bladder specimens were obtained from Sun Yat-sen Memorial Hospital (SYMH). Cohort 1 included 210 BCa tissue specimens and 59 normal urothelial tissues, while Cohort 2 consisted of 59 paired microarray tissues, both of which were obtained from Sun Yat‐sen University Cancer Center between 2000 and 2013 (Supplementary Table UBE2S expression was classified as low (H-score <100) or high (H-score ≥100). LPP expression was classed as low (H-score <70) or high (H-score ≥70).The IHC procedures and analysis were conducted as previously described . The anthttps://cancergenome.nih.gov/) and Gene Expression Omnibus  . Ubiquitination-related genes were downloaded from the Molecular Signature Database : KEGG UBIQUITIN MEDIATED PROTEOLYSIS. The volcano plot and Venn diagram were drawn using R software.The omics data were obtained from two platforms: The Cancer Genome Atlas  were purchased from the American Type Culture Collection  and cultured as previously described . More deUBE2S, LPP, and TRIM21 and the negative control siRNA (si-Ctrl) were purchased from GenePharma   oligonucleotides targeting a) Table . The siRUBE2S were cloned into the pLKO.1-Puro vector, while full-length UBE2S, TRIM21 and LPP were cloned into the pCDH-CMV-MCS-EF1-Puro vector. All vectors were purchased from IGE Biotechnology , and their sequences are listed in Table Short hairpin RNA (shRNA) sequences targeting −ΔΔCt method  was used to calculate relative expression. All qPCR primers are listed in Supplementary Table Total RNA was extracted from fresh tissues with TRIzol Reagent  or from cells with an RNA Quick Purification kit . RNA (1 μg) was reverse transcribed to complementary DNA templates using a reverse transcription kit . qPCR was conducted using a Light Cycler 480 system  with SYBR Green reaction mix . The 2All proteins were extracted using RIPA lysis buffer supplemented with protease inhibitors  and quantified by a BCA Protein Assay Kit . After being electrophoresed, transferred and blocked with 5% skim milk, proteins were incubated with primary and secondary antibodies and visualized using ultrasignal chemiluminescence . The antibodies are listed in Supplementary Table All these experiments were performed as described previously . Photos 6 UM-UC-3 cells with stable expression of firefly luciferase were inoculated into the footpads of randomized male BALB/c nude mice . Usually, all mice were euthanized using cervical dislocation at the 4th week in accordance with the Declaration of Helsinki. The volume of LNs was calculated as follows: LN volume (mm3) = length × width2 × 0.5. Cephalomannine  was dissolved in 5% dimethylacetamide (DMSO), 40% PEG300, 5% TWEEN80 and 50% distilled water. Cephalomannine (200 μl) was injected intraperitoneally into the therapeutic groups, and PBS was used as the control. No statistical method was used to predetermine the sample size for the mouse experiment, which was based on previous experimental observations. The investigator was blinded to the group allocation of the animals during the experiment.All animal experiments were approved by the Sun Yat-sen University Institutional Animal Care and Use Committee (SYSU-IACUC-2023-000232). The procedures were introduced previously . BrieflyImmunofluorescence staining assays were performed as previously described . More deUBE2S, TRIM21, LPP, TRIP13, PRMT1, UBR5 and UBE3C. Briefly, cell lysates were incubated with 2 μg of the corresponding antibodies , UBE2S , TRIM21 , LPP  and ubiquitin (Ag20753 Proteintech), were purchased, while ubiquitin-K11R protein was expressed by a cell-free protein expression kit  according to the manufacturer’s instructions. This assay was performed according to the report of Jixi et al. [LPP, 1 μg UBE1, 1 μg UBE2S, 1 μg TRIM21, 5 mM ATP  and 5 mM MgCl2  was incubated at 37 °C for 1 h. Reactions were terminated by adding 4× loading buffer and subsequently analyzed by western blot with an antibody against LPP .Recombinant proteins, including i et al. . BrieflyFour fresh BCa tissues were acquired with informed consent and transported in tissue storage solution . The clinical information of the patients is described in Supplementary Table t test was used to analyze the differences between two groups, whereas one-way or two-way analysis of variance (ANOVA) was performed to compare the differences among at least three groups. All statistical assessments and plotting were executed using Statistical Product and Service Solutions  and GraphPad Prism 7.0 software. Statistical significance was considered as *P < 0.05, **P < 0.01.Data from at least three independent experiments are presented as the means ± standard deviations (means ± SDs). Unpaired or paired Student’s two-tailed UBE2S was one of the most significantly upregulated genes in both BCa tissues with lymphatic metastasis (LN+ CA) and metastatic LNs (LN+) compared with normal adjacent tissues (NAT)  Fig. . FurtherUBE2S protein levels in cohort 1 using IHC assays and found that UBE2S, which localized in both the nucleus and cytoplasm, was nearly absent in NAT and exhibited stepwise increases from nonmuscle-invasive bladder cancer (NMIBC) to muscle-invasive bladder cancer (MIBC) without lymphatic metastasis (LN− MIBC) to MIBC with lymphatic metastasis (LN+ MIBC)  in BCa patients compared to those without metastasis (M-) in both TCGA and GSE48277 databases   Fig. . Consistase Fig. , and in ase Fig. . In addises Fig. . We alsoets Fig.  but alsoets Fig. . SimilarUBE2S expression experienced both lower overall survival (OS) and disease-free survival (DFS) rates than those with low UBE2S expression in cohort 1. Further univariate and multivariate Cox regression analyses confirmed high UBE2S expression as a risk factor for both the OS (P < 0.001) and DFS (P < 0.001) of BCa patients  for further study from 27 potential substrate proteins of UBE2S  and mass spectrometry assays of wild-type T24 and UM-UC-3 cells with a tastasis –35. In alls Fig. .Fig. 3UBLPP protein was increased in UBE2S-knockdown BCa cells but decreased in UBE2S-overexpressing cells, while TRIM21 expression was not affected , indicating that UBE2S mediated LPP degradation via ubiquitination. Previous studies reported that K11-linked polyubiquitination by UBE2S or other ubiquitin-related enzymes mediated the degradation of various substrate proteins [UBE2S markedly decreased both the total and K11-polyubiquitination but not K48- or K63-linked polyubiquitination of MYC-tagged-LPP, which was more obvious in TRIM21-knockdown HEK293T cells   Fig. , which sTRIM21 contains four domains, including the RING-finger, B-box (BBOX), coiled-coil and PRY-SPRY domains [TRIM21 is responsible for the function of its E3 ligase [TRIM21 that lacked the RING domain (ΔRING)  Fig.  and founNG) Fig. , which cUBE2S–LPP regulation in BCa patients. As displayed in Fig. UBE2S and LPP . Chi-square tests indicated that LPP expression was negatively related to N and M stages  Fig. . Functioids Fig. . Taken tUBE2S that is involved in the LN metastasis of BCa. Mechanistically, UBE2S directly interacted with TRIM21 to enhance the metastatic capacities and EMT of BCa cells by degrading LPP via K11-linked ubiquitination. Furthermore, targeting UBE2S with cephalomannine was a promising strategy to prevent the lymphatic metastasis of BCa cells , which affects nearly all pivotal functions in tumor cells, is crucial for maintaining protein homeostasis . As partLPP), a member of the zyxin family of LIM proteins, functions as an actin-binding protein and transcriptional coactivator and plays an essential role in metastatic processes [LPP transcriptionally regulates ETV5 to activate Zeb1, which later suppresses E-cadherin to promote EMT [LPP directly regulates MMP15 to degrade N-cadherin, which results in inhibition of EMT in lung cancer [LPP suppressed metastasis-related phenotypes and EMT and served as a tumor suppressor in BCa. Furthermore, knockdown of LPP effectively rescued the inhibition of metastasis in UBE2S-silenced cells, highlighting that UBE2S regulated the malignant progression of BCa in an LPP-dependent manner.Lipoma preferred partner (rocesses . Howevermote EMT . In contg cancer . In our TRIM21) was first discovered for its antiviral function in innate immunity but has recently attracted increasing attention in cancer metabolism and tumorigenesis [TRIM21 regulates multiple substrates through different types of ubiquitination, among which K11, K27, K33, K48 and K63 have been reported [UBE2S transferred Ub to TRIM21 and cooperatively formed K11-linked ubiquitin chains to mediate the degradation of LPP in BCa.Tripartite motif containing 21 , carfilzomib (Kyprolis), and ixazomib (Ninlaro), have become foundational chemotherapeutic drugs in the clinical treatment of multiple myeloma and mantle cell lymphoma . In the e cancer , 21. OveUBE2S in the lymphatic metastasis of BCa and clarifies the UBE2S-TRIM21-LPP axis as a novel proteasome-mediated mechanism. Cephalomannine is a promising treatment targeting UBE2S and serves as an effective drug for metastatic BCa.In summary, our work provides evidence for the crucial roles of Supplemental materialsaj-checklistOriginal Data File


Here we suggesta correction for the widely used AMBER OL3 ff byadding a simple adjustment of the nonbonded parameters. The reparameterizationof the Lennard–Jones potential for the −H8···O5′–and −H6···O5′– atom pairs addressesan intranucleotide steric clash occurring in the type 0 base-phosphateinteraction (0BPh). The nonbonded fix (NBfix) modification of 0BPhinteractions (NBfix0BPh modification) was tuned via a reweightingapproach and subsequently tested using an extensive set of standardand enhanced sampling simulations of both unstructured and foldedRNA motifs. The modification corrects minor but visible intranucleotideclash for the anti nucleobase conformation. We observedthat structural ensembles of small RNA benchmark motifs simulatedwith the NBfix0BPh modification provide better agreementwith experiments. No side effects of the modification were observedin standard simulations of larger structured RNA motifs. We suggestthat the combination of OL3 RNA ff and NBfix0BPh modification is a viable option to improve RNA MD simulations.Molecular dynamics(MD) simulations represent an establishedtoolto study RNA molecules. The outcome of MD studies depends, however,on the quality of the force field ( The general usage, applicability, and reproducibilityof MD simulations are continuously increasing. However, the MD simulationstudies remain limited by the approximative nature of the ffs.Insights into RNA structural dynamicsat an atomistic descriptionare essential for the understanding of biomolecular motions and processes.Molecular dynamics (MD) simulation is an established theoretical toolthat benefits from the ability to overcome experimental limits whenfinding links between the RNA structure, dynamics, and function.ffs are presentlyavailable tocarry out MD simulations of RNA systems,11 but none of them is flawless.18 Despite the suboptimal performance for certain motifs, mainly theshort RNA single strands,21 the AMBER OL3 ff from 20107 still remains the state-of-the-art for RNA simulationsand safest option to start with.22 It hasbeen shown in the past decade that the OL3 performance can be partiallytweaked by optimizing simulation settings and by adding new ff terms that are orthogonal to the current ff terms; namely, (i) an update of parameters for phosphate oxygensby Steinbrecher and co-workers23 was shownto improve the general simulation outcome,25 (ii) the combination of OL3 with the four-point OPC water model26 revealed benefits for structural descriptionof short RNA single-strands  and tetraloops(TLs),27 (iii) controllablefine-tuning of specific pairwise H-bond interactions via an externalgeneral H-bond fix potential (gHBfix) corrected structural ensemblesof several RNA motifs,28 and (iv) specific adjustment of interactions formed by terminalnucleotides via tHBfix further improved the agreement with experimentsfor RNA TNs.29A number of RNA 30 we spotted anotherminor but visible imbalance of the AMBER ff connectedwith the imprecise description of RNA intranucleotide interactionswhen van der Waals (vdW) clash in GS+1(C8–H8)···GS+1(O5′) interaction resulted in the spurious flippingof the GS+1 sugar–phosphate backbone and populationof non-native structures of UUCG TL. The identified vdW clash in UUCGTL30 is related to the weak −CH···O–H-bond between −C8H8 and −C6H6 groups of purines andpyrimidines, respectively, and bridging O5′ phosphate oxygenfrom the same residue, which was categorized as base-phosphate interactiontype 0  stabilizedthe native GS+1 phosphate conformation30 and indicated that it could be beneficial for the stabilityof native states of both GAGA and UUCG TLs.32In our recent work, 0 0BPh, 1.31 The 0BPh modification)as a general improvement of the AMBER OL3 RNA ff . We first used the reweighting approach to optimizesettings for NBfix0BPh modification. Reweighting methodsare one of the strongest tools of ff development33 and can re-evaluate the results of MD simulations under the assumptionof a modified ff parametrization without repeatingthe entire simulation. Reweighting allowed us to identify the mostsuitable modified parameters for both purines and pyrimidines, whichwere subsequently validated on benchmark systems through large-scaleMD simulations. The testing set contained single-stranded RNAs, duplexes,TLs, and some examples of more structured RNA motifs with noncanonicalinteractions. We also investigated possible effects of the NBfix0BPh modification (with a similar setting as those for RNAnucleotides) on DNA duplex and guanine quadruplexes (GQs). We showthat the NBfix0BPh modification improves the structuralbehavior of small RNA motifs by increasing the agreement with experimentaldata sets. The stability of canonical A-RNA duplexes was also improved,whereas other bigger and more structured RNA motifs were not affectedby the NBfix0BPh modification on the affordable simulationtime scale. In addition, this paper presents an extensive set of newsimulations with two versions of the external gHBfix potential andsupports previous claims28 that gHBfix is beneficialfor proper structural description of RNA motifs.In this work, we performed an extensive set ofstandard and enhancedsampling MD simulations on diverse RNA systems and considered theadjustment of 0BPh interaction via NBfix  modificationof phosphate oxygens developed by Steinbrecher et al.,23 where the affected dihedrals were adjusted asdescribed elsewhere.24 This RNA ff versionis abbreviated as OL3CP henceforth, and the AMBER libraryfile can be found in the Supporting Information of ref , where all RNA H-bond donor···H-bondacceptor interactions are modified; i.e., base donor–base acceptor,base donor–sugar acceptor, base donor–phosphate acceptor,sugar donor–base acceptor, sugar donor–sugar acceptor,and sugar donor–phosphate acceptor are adjusted specifically and older ff99bsc036 RNA ffs.We used standard AMBER OL3 n of ref . In addi for the – H8···O5′–and −H6···O5′– pairs, i.e., betweenH5–OR/OS and H4–OR/OS atom types, by 0.25 Å to2.8808 and 2.9308 Å for purine and pyrimidine nucleotides, respectively. The OR atom type belongs to O5′and O3′ bridging phosphate oxygens in the RNA OL3CP, whereas the OS atom type involves O5′, O3′, and alsoO4’ atoms from deoxyribose sugar in the DNA OL15 ff. Depths of the potential well  were kept at a default value of 0.0505 kcal/mol. Inother words, we attempted to decrease the repulsion between H8 andH6 atoms of all purine and pyrimidine bases and O5′ oxygensof phosphates. We note that O3′ and O4’ atoms have higherdistances from H8 and H6 in comparison with O5′ atoms, andthus, modified Lennard–Jones potentials for H8/H6···O3′/O4’atom pairs are supposed to have marginal effects.On top of that,we modified the pairwise vdW parameters via breakage of the combination(mixing) rules via the nonbonded fix (NBfix) approach40 as one strand of an A-formduplex. RNA single strands were solvated using a cubic box of theOPC water26 with a minimum distance betweenbox walls and solute of 12 Å, yielding ∼2200 water moleculesadded (∼40 × 40 × 40 Å3 box size),∼4100 water molecules added (∼50 × 50 × 50Å3 box size), and ∼7600 water molecules added(∼60 × 60 × 60 Å3 box size) for TNs,HNs, and the GAGA TL, respectively. Enhanced sampling MD simulationswere performed in ∼0.15 M KCl salt excess (TNs and HNs) and∼1.0 M KCl salt excess (GAGA TL) using Joung–Cheatham(JC) ionic parameters41 for the TIP4P-EWwater.Initial coordinatesof r(AAAA), r(CAAU), r(CCCC), r(GACC), and r(UUUU) tetranucleotides(TNs); r(UCAAUC) and r(UCUCGU) hexanucleotides (HNs); and r(gcGAGAgc)8-mer tetraloop (GAGA TL) were prepared using the Nucleic Acid Builderof AmberTools141QC0;42 only 10 canonical base pairs wereconsidered, and PDB ID 1RNA43), RNA sarcin–ricinloop , RNA kink-turn7 ,DNA duplex  with a minimum distance between boxwalls and solute of 10 Å and with ∼0.15 M KCl salt usingthe JC ionic parameters. The TIP3P water model51 was also used for some specific tests involving RNA duplexes(Table S2 in the Supporting Information).Starting topologies and coordinates of two RNA duplexes(PDB ID B ID 1BNA), and tw1BNA(1KF1 and (3 +1KF1(2GKU) were prn of ref  for detaT = 298K withthe hydrogen mass repartitioning52 allowinga 4 fs integration time step (see the Supporting Information of ref (53 whereas both AMBER18and GROMACS201854 were used for enhancedsampling simulations. PARMED55 was usedto convert AMBER topologies and coordinates into GROMACS inputs.All MD simulations were run at n of ref  for othe56 and well-temperedmetadynamics59 (MetaD) in combination with the REST2 method (ST-MetaD).60 REST2 simulations were performed at 298 K (the reference replica)with 8 and 12 replicas for TNs and HNs, respectively. The scalingfactor (λ) values ranged from 1 to 0.601700871 and to 0.59984for 8 and 12 replicas, respectively. Those values were chosen to maintainan exchange rate above 20%. The effective solute temperature rangedfrom 298 to ∼500 K. REST2 simulations were performed with theAMBER GPU MD simulation engine (pmemd.cuda).61 Further details about REST2 settings can be found elsewhere.14 One test simulation of r(UCUCGU) HN was performedat 275 K temperature corresponding to the experimental conditionswhere the biggest number of NMR signals was obtained.16 The same λ values were applied for scaling, whichresulted in an effective solute temperature range from 275 to ∼460K. We note that the 275 and 298 K REST2 simulations revealed comparableresults considering the limits of sampling.We used two differentenhanced sampling schemes, i.e., a standard replica exchange solutetempering (REST2) protocol62 was used as a biased collective variable.63 ST-MetaD simulations were carried out using a GPU-capable versionof GROMACS201854 in combination with PLUMED2.565 (see ref (Table S2 fora full list of standard as well as enhanced sampling simulations).ST-MetaD simulationsof GAGA TL were performed with 12 replicas starting from unfoldedsingle strands and were simulated in the effective temperature rangeof 298–497 K for 5 μs per replica. The average acceptancerate was ∼30%. The εRMSD metric backbone 3J scalar couplings,(ii) sugar 3J scalar couplings, (iii) nuclear Overhausereffect intensities (NOEs), and (iv) the absence of specific peaksin NOE spectroscopy (uNOEs). In addition, we also considered ambiguousNOEs (ambNOEs) resulting from the sum of overlapping peaks21 as the fifth individual NMR component for HNs. 3J scalarcouplings were calculated via Karplus relationships; NOEs and uNOEswere obtained as averages over the N samples, i.e., N samples, i.e., 2 value for each REST2 simulation: i2 and ni are individualχ2 valuesand the number of observables, respectively, corresponding to theparticular NMR observables. The lower the total χ2 value is, the better is the agreement between the experiment andREST2 simulation G°fold).32 The reference native structure of GAGA TL was taken from our previouswork.14 The εRMSD threshold separatingthe folded and un(mis)folded states was set at a value of 0.7 (seeref (G°fold estimations andconvergence).ST-MetaD simulation of the GAGA TLprovided populations of the native structure and other conformations,which can be used for estimation of the folding free energy balance, r(CCCC),and r(CAAU) TNs29 and from folding OL3CP ST-MetaD simulations with gHBfix19 of GAGA and UUCG TLs.32 We monitored changes in total χ2 values (and its components) and ΔG°fold energies for TNs and TLs, respectively. Each NBfix0BPh setting was defined by scanning Ri,j values of the Lennard–Jones potentialfor the −H8···O5′– and −H6···O5′–atom pairs from 2.33–3.33 and 2.48–3.48 intervals forpurine and pyrimidine nucleotides, respectively, using 0.025 Åsteps.The reweightingalgorithmRi,j parametersfor both purine and pyrimidine nucleotides is optimal . Reweighted data from r(CCCC) and r(CAAU) simulations with thesole gHBfix19 suggest an even larger decrease  of the Ri,j value for pyrimidines, i.e., −H6···O5′–atompairs, but that is contradicted by data from simulations with combinedgHBfix19 + tHBfix20 potentials . Data fromthe reweighting of ST-MetaD TL simulations support results from TNs.Reweighting of the GAGA TL simulation shows that the adjustment of Ri,j values for the −H8···O5′–atom pairs of purine nucleotides is the dominant contributor for shiftingΔG°fold energy up to ∼3.5 kcal/mol   is sampling noncanonical anti orientation of the χ dihedral  2B. In co UUCG TL 2B.Ri,j parametersfor both −H8···O5′– and −H6···O5′–atom pairs provided better agreement with experiments. Effective usageof reweighting requires a broad exploration of the conformationalspace, which we believe was achieved in trajectories from REST2 andST-MetaD simulations of TNs and TLs, respectively. The accuracy ofreweighted results decreases with increasing deviation of the parameterfrom original values  and explicitly testedits effect on the structural description of five RNA TNs and two HNs,i.e., benchmark set of motifs with available NMR data, in a largeset of enhanced sampling simulations. We again opted for the REST2protocol, which was shown to provide ensembles with sufficient convergencefor those single-stranded motifs.29 We used OL3CPff combined with gHBfix19, gHBfix19 + tHBfix20,and gHBfix21 + tHBfix20 potentials, which were shown to improve thestructural behavior of the short RNA single strand motifs (see Table S2 in Supporting Information). The data are summarized in We used the optimized setting for the NBfix0BPh modificationfurther fine-tunesespeciallythe r(CAAU) and r(AAAA) ensembles, which now agree excellently withNMR data  TN. We did observe an increase (∼20%)in the population of the canonical A-form like structures (as forother TNs), but it was associated with a slight increase of totalχ2 values; i.e., the agreement betweensimulations and experiment was modestly worsened for all tested MDsetups  1-3_2-4 stacked conformers29 and supported the sampling of unstructured statesin structural ensembles, which resulted in excellent agreement withthe NMR data.17 STAfix, however, is notsuitable for simulations of folded RNAs; it is a special-purpose modificationthat has been prepared for binding of unstructured single-strandedRNAs to proteins. We also note that we have significantly fewer NMRsignals for r(UUUU) than for other TNs, which complicate predictionsfrom the experiment.Recently, modification of the AMBER potential(named STAfix) bymajor weakening of intramolecular stacking was employed to study thespontaneous binding of RNA single strands to RNA recognition motifproteins.0BPh modification improvesthe structural behaviorof two r(UCAAUC) and r(UCUCGU) HN motifs. It has been shown previouslyby both experiments and simulations that r(UCAAUC) should sample predominantlystructures around the canonical A-form state.76 On the other hand, the presence of the G residue in r(UCUCGU) HNsignificantly affects the conformational space, and this HN motifis much more dynamical than shorter RNA TNs.16 r(UCAAUC) REST2 simulations performed here show that the NBfix0BPh modification slightly enhanced the population of canonicalA-form like states for r(UCAAUC) HN, and results are in good agreementwith the experiment21  structural ensembles, we performedsimulations with only four different setups for this motif  TN and especially r(UCUCGU) HN motifsremain challenging for the AMBER OL3CPff even with the additional refinements. Despite some uncertainty inexperimental data sets for those motifs, it is evident that a largerreparameterization than a simple adjustment of pairwise terms forone interaction would be required to improve the structural behaviorof those motifs. Taking all of the available data together clearlydemonstrates that we remain far from having flawless ff for RNA molecules.In summary, the NBfix16 We observed that calculated χ2 values are almost an order of magnitude higher than those from TNsimulations and the other r(UCAAUC) HN .Analysis of most populated conformers during REST2 simulation showedthat besides the canonical A-form-like states, MD favors formationof a looplike conformer with formation of G5C2 base pair . The experimentshows much weaker NMR A-form signals than for TN and r(UCAAUC) HNmotifs, which indicates the presence of another state.16 However, the formation of conformers with theG5C2 base pair is not supported.16r(UCUCGU) REST2 simulations revealeda striking disagreement betweenMD predictions and the experimental data.AAUC) HN 1 and 2. ents see  revealedCPff simulationcould also (to some extent) affect the structural ensemble of ther(UCUCGU) HN. This is out of the scope of this work and will be addressedin the future.In summary, r(UCUCGU) NMR data are composed of different conformers,but those sampled during REST2 simulations  are apparently different from those detected inthe experiment. We speculate that a different preference for the C2′-endosugar pucker in the AMBER OL30BPh modificationon the folding of this small 8-mer RNA TL motif. We obtained a ΔG°fold energy of −0.5 ± 0.6kcal/mol (corresponding to 68.3 ± 20.0% of the population ofthe native state). The result is in an excellent agreement with datafrom the reweighting approach (see 78), the agreement between predicted data from reweightingand the actual simulation result is encouraging. The GAGA native stateis stabilized by ∼0.6 kcal/mol in comparison with the controlsimulation ,32 indicating that the NBfix0BPh modification could increase the population of the GAGAnative state by ∼24% at 298 K.We performed ST-MetaDsimulation of the r(gcGAGAgc)TL to explicitly test the effect of the NBfixoach see ). Despit0BPh modification on the structuraldescription of small RNA motifs, one should test its effects on biggerand structured RNA and DNA motifs before any claims are made aboutits general applicability. Hence, we performed standard MD simulationsof systems commonly used for NA ff validation, i.e.,RNA and DNA duplexes, RNA SRL and Kt-7 motifs, and two DNA GQs. Wecompared the results with the control simulation set .Despite theclear benefits of the NBfix0BPh supports canonical A-form backbone conformation in shortRNA single strands. In addition, simulations of RNA duplexes revealedthat, similarly to the OL3 correction,79 NBfix0BPh modestly reduces the inclination of the A-formduplex (see the Supporting Information).Therefore, we also tested the possible effect of NBfix0BPh on the potential formation of a spurious deformed duplex structureknown as the ladderlike RNA structure,80 which is associated with the population of high-anti χ angle in RNA duplexes. Elimination of the ladderlike structurehas been the key improvement introduced by the OL3 parametrization.7 Thus, we tested NBfix0BPh on a longer1QC0 duplex42  sequence, see 2 duplex known for a quick (∼10ns) transition from the canonical A-form to the spurious ladderlikestructure in obsolete AMBER ff versions lacking theOL3 correction.79 We compared the behaviorof two ffs, i.e., the outdated ff99bsc036 and the currently recommendedOL3 ff, in combination with two different water models(TIP3P51 and OPC26) and explicitly probed possible effects of adding the NBfix0BPh modification. As a result, we compared the behavior ofeight different simulation setups (Table S4 in Supporting Information) on both duplexes. The initial set ofr(CGCG)2 simulations revealed a high tendency to end fraying,i.e., loss of terminal base pairs, which significantly affected outcomesof the simulations (Table S4 in Supporting Information). Therefore, we used the gHBfix21 potential28 in the next set of r(CGCG)2 simulations to stabilizebase–base interactions and suppress the extensive base pairfraying. We observed that a spontaneous transition into the ladderstructure occurred only in the ff99bsc0 + TIP3P setupfor both short and long duplexes (Table S4 in the Supporting Information). Thus, besides the known reparameterizationof χ dihedrals of nucleosides in the OL3 ff,7 there might be other factors affectingthe propensity of transitions of canonical duplexes to ladders, namely,(i) the four-point OPC water model and (ii) the NBfix0BPh modification . Subsequently, we started simulationsfrom the ladder conformation of the r(CGCG)2 duplex andobserved that only setups with OL3 ff were able toundergo successful transitions back to the canonical A-form structureson the 1 μs time scale; the ladders were corrected in all fourOL3 simulations on time scales ∼100–250 ns. NBfix0BPh alone did not eliminate the ladder, at least on the 1μs time scale (Table S4 in Supporting Information).We showed in a previous section that NBfix0BPh modification is a vitaladdition to the standard AMBER OL3 RNA ff. It canfurther stabilize canonical A-form duplexes and prevent transitionsto artificial ladders. Results from duplex simulations revealed that anti/high-anti χ imbalance of nucleosides was not the only driving factor for spurious transitions into ladderlikestructures. The clash in the backbone remained30 even after applying OL3 correction, and this clash canbe efficiently removed by the NBfix0BPh modification. TheOL3 ff can be safely combined with NBfix0BPh, and this combination further reduces the likelihood of samplingvarious spurious structures.In summary, the NBfixff for atoms involved in the0BPh intranucleotide molecular interactions, i.e., the NBfix0BPh modification. This was motivated by the recently identified spurioussteric clash in the sugar–phosphate backbone associated withthe 0BPh interaction.30 We optimized NBfix0BPh settings by the reweighting method, which is an efficientapproach to assess the effects of ff changes withoutthe necessity to perform actual extensive simulations. Reweighteddata indicate that the 0.25 Å decrease of pairwise Ri,j parameters for both −H8···O5′–and −H6···O5′– atom pairs is theoptimal adjustment.We presented the adjustment of pairwiseLennard–Jones parametersof the OL3 AMBER RNA 0BPh modification wasfurther verified by anextensive set of new enhanced sampling simulations for a contemporaryrecommended set of benchmark RNA systems including tetranucleotides,hexanucleotides, and tetraloops. The simulations with the NBfix0BPh modification provided better agreement with the availableexperimental data. We suggest that the NBfix0BPh modificationshould be used in all future simulations of these small RNA oligonucleotidesbecause it directly corrects a real imbalance of the AMBER ff nonbonded terms. Elimination of its consequences by other ff terms, such as dihedral potential reparameterizationsor gHBfix, is thus not desirable. We also did not find any undesiredside effects of the NBfix0BPh modification in standardsimulations of larger and more complicated RNA motifs with noncanonicalinteractions. The data for canonical duplexes indicate that NBfix0BPh may help to prevent A-RNA distortions into the ladderlikestructure, although the OL3 still remains essential in preventingthis artifact.The NBfix0BPh modificationcould be consideredas a general addition to the OL3 RNA ff . NBfix0BPh eliminates artificialsteric clash within nucleotides sampling the anti nucleobase conformation, which improves the description of syn/anti balance. Considering the resultsshown here, we suggest the utilization of the NBfix0BPh modification on top of the AMBER OL3 ff,7 modified parameters for phosphates by Steinbrecheret al.,23 OPC water model,26 and the gHBfix21 potential28 for MD simulations of RNA especially for small motifs such as shortsingle strands and tetraloops.Hence, the NBfix0BPh modification corrects a moderatebut evident imbalance in the repulsion term of the AMBER ff nonbonded terms for the 0BPh interaction, which affects the accuracymainly in case of the simulations of small RNA oligonucleotides. Asthe NBfix0BPh modification selectively targets only thesolute–solute van der Waals term related to the 0BPh interaction,it is robust and does not produce any side effects. Thus, it couldbe used in combination with any ff from the AMBERfamily.In summary, NBfix


Frontiers in Cell and Developmental Biology  and reverse transcription–polymerase chain reaction and/or immunohistochemical (IHC) analysis; ii) availability of histopathology, imaging, and clinical information; iii) exclusion of the possibility of bone metastasis  and the triple sign and pseudo-cystic sign (via magnetic resonance imaging), those results had relatively low sensitivity  precise diagnosis, ii) adequate clinical information, and iii) exclusion of metastasis. Such reviews would enable clarification of the real clinicopathological characteristics of the disease and, in turn, lead to meaningful prospective studies for standardization of therapy.