Frontiers in Molecular Biosciences

In our past study on human cilia from primary ciliary dyskinesia (PCD) patients using cryo-electron tomography and mass spectrometry, defects in the DNAH5 gene were proven to cause the loss of the entire outer dynein arm along with other proteins. This phenomenon was not observed in the unicellular green alga Chlamydomonas. In this study, we examined the loss of ciliary components caused by defects in various genes, based on proteomic and structural analyses of motile cilia from multiple PCD patients. The absence of single outer arm dynein genes causes a loss or significant decrease in other inner and outer arm dyneins. Interestingly, some patients with outer dynein defects show decreased intraflagellar transport (IFT) proteins, suggesting an influence of cargo components or assembly on transport. These phenomena were not observed in Chlamydomonas, suggesting a more complex mechanism of ciliogenesis in humans despite the high similarity in the final 3D architecture of the 9+2 axoneme. Defects in certain central pair proteins unexpectedly reduced components on the doublets as well. Our results demonstrate protein-protein interactions during stages of human motile ciliogenesis that are more complex than those in unicellular organisms.

Oncogenic mutations are known to occur at non-random rates and specific hotspots, yet the structural and thermodynamic factors underlying these hotspots remain poorly understood. This study presents molecular dynamics simulations of 22 cancer hotspots across 10 oncogene families, simulated as histone-DNA complexes. Across 18 of 22 mutations, thermodynamic effects were predominantly localized to within 6 [A] of the DNA-histone interface (mean capture 104%), with van der Waals interactions driving the effect at the thermodynamic extremes, indicating that oncogenic mutations alter nucleosome binding through precise local contact changes rather than global structural rearrangements. Further, analysis revealed a gene-family correlated pattern in thermodynamic stability. RAS family mutations showed a consistent trend toward nucleosome stabilization (mean {Delta}{Delta}G = -4.82 kcal mol-1), while kinase domain mutations trended toward destabilization (mean {Delta}{Delta}G = +40.12 kcal mol-1), a difference reaching statistical significance in this exploratory analysis (Mann-Whitney U, p = 0.008). This interface-specific mechanism, combined with the gene-family-correlated thermodynamic pattern, provides a biophysical framework for understanding nucleosome-level contributions to cancer hotspot mutation biology.

P-type ATPases maintain cellular metal homeostasis by coupling ATP hydrolysis to the active translocation of ions across biological membranes. Within this superfamily, P1B -type ATPases play a central role in the transport and detoxification of transition metals such as Cu+ and Ag+. Despite the availability of several crystallographic structures for bacterial ATPases, molecular determinants governing ion selectivity, coordination dynamics, and transport efficiency are not yet fully resolved, particularly for plant homologs, whose conformational landscapes have not been explored in detail at atomistic resolution. In this study, we combine molecular dynamics (MD) simulations with enhanced sampling through metadynamics to characterize the conformational dynamics and ion-binding mechanisms of representative heavy-metal P-type ATPases. By comparing bacterial and plant homologs in phosphorylated and dephosphorylated states, we examine how phosphorylation is associated with changes in structural plasticity, channel accessibility and ion coordination environments. The simulations indicate phosphorylation-dependent rearrangements of the cytosolic domains and transmembrane helices, which may alter access to the metal-binding pathway. Enhanced sampling identifies distinct conformational basins and possible transition pathways along functionally relevant collective variables, allowing characterization of the free-energy landscape associated with ion displacement within the transmembrane region. Together, these results provide an atomistic perspective on the relationship between metal coordination and protein conformational dynamics. Overall, our findings support a model in which phosphorylation state, structural dynamics, and local coordination environments collectively contribute to the differential behavior of metal ions in P1B -type ATPases.

Translation initiation is a critical stage of protein synthesis in prokaryotes, where ribosomes assemble on mRNA to begin decoding genetic information. The interactions between ribosomal RNA (rRNA) and mRNA significantly influence this process, with features like ribosomal binding site (RBS) affecting ribosomal dynamics. However, direct experimental validation of these types of interaction is yet to be provided. In this study, we developed an optimized small sub-unit sequencing (SSU-seq) protocol for Escherichia coli, enabling the detailed examination of ribosomal complexes and subunits dynamics. Unlike Ribo-seq, which primarily maps fully assembled ribosomes, SSU-seq captures both ribosomal subunits and intact ribosomes, offering a comprehensive view of translation initiation and elongation events. By integrating experimental data with predictive models, our study advances the understanding of translation regulation in prokaryotes and underscores the utility of SSU-seq in decoding the complexities of ribosome-mRNA interactions. Our analysis reveals distinct patterns of small subunit distribution across the entire mRNAs with higher density near positions with stronger rRNA-mRNA hybridization. The results may suggest that rRNA-mRNA hybridization positions along the entire transcript facilitate efficient start codon recognition by maintaining proximity between the small subunit and the mRNA.

Carbohydrates are essential molecules involved in a wide range of biological processes. While their structural representation in the Protein Data Bank (PDB) has been hindered due to their highly diverse and flexible nature, advancements on X-ray crystallography and CryoEM are accelerating the obtaining of their experimental structures, which in turn supports improved analyses of their structural properties, such as the role of their non-chair conformations on biological processes. In this context, the current work provides a comprehensive and up to date overview of carbohydrate structures within the PDB through classification, quantification, and characterization of monosaccharides and glycan chains, incorporating Cremer-Pople puckering coordinates to evaluate pyranose conformations. Our findings show that, even with a steep increase in CryoEM structures including more complex oligosaccharides, carbohydrate data remains concentrate dinsmallglycanchains, with a about 4% of non-chair conformation sintopresolution structures. Free energy landscapes for the pyranose ring in the presence and absence of nine different binding proteins, including both enzymes and non-enzymes, reinforce the preferred stability of chairs and the rarity of conformational induction upon binding.

Rule-based languages have proven effective for modeling systems of interacting structured entities as typically encountered in chemistry and molecular biology. We present PyKappa, a rule-based modeling package written in Python whose interpreted nature enables interactive simulation and analysis, including by agentic AI. The package seeks to broaden the base of developers by utilizing a widely known programming language and serves as an easy-to-deploy teaching tool. Using PyKappa, we conduct a case study of phase separation.

Background: Comprehensive evaluations of lung nodule detection methods remain limited, particularly regarding data from clinical routine, novel photon-counting CT (PCCT) technology, and the comparison of academic and commercial models. Materials and Methods: This retrospective study collected 1860 routine PCCT scans acquired at Hannover Medical School from 2021 to 2024. We compared four deep-learning-based lung nodule detection models, which were developed for conventional CT, namely the publicly available TotalSegmentator, nnDetection, and RadYOLO methods, as well as a commercial lung computer-aided detection (CAD) system. We evaluated the inter-model agreement in an unannotated cohort (n=1699), sensitivity in a smaller cohort of 25 manually annotated scans, and false positives as detections in 136 report-negative scans. Results: In the unannotated cohort, the commercial CAD system detected 7071, TotalSegmentator 4264, nnDetection 5254, and RadYOLO 3122 nodules. Overall, the inter-model agreement was low, and the models produced substantially different candidate nodules. Approximately 1300 nodules were detected by all models, representing 18.7% - 42.8% of each model's detections. 15.4% - 37.4% of a model's detections did not match any other model. The models showed moderate sensitivity (between 0.71, 95%-CI [0.64, 0.77], and 0.83, 95%-CI [0.77, 0.88]), and TotalSegmentator and RadYOLO produced few false positives per scan (both 0.85, 95%-CI [0.70, 1.02]). Conclusion: The lung nodules detected by four deep learning models showed high variation and a lack of consensus on clinical PCCT data. These findings highlight the importance of model benchmarking and local validation when deploying models in real-world clinical workflows.

Amyloid {beta}-protein (A{beta}), which is known for its role in Alzheimer's disease (AD), is also involved in several physiological functions. Of the two predominant isoforms, A{beta}40 and A{beta}42, the latter is more aggregation-prone and more active in health and disease. In the brain, A{beta} interacts with free lipids, which may affect its assembly pathways. Here, thioflavin T (ThT) fluorescence spectroscopy is combined with molecular dynamics (MD) to investigate A{beta}40 and A{beta}42 co-assembly with free dimyristoylphosphatidylcholine (DMPC) and DMPC/cholesterol (CHOL) lipids. ThT measurements at several protein-to-lipid (PL) ratios show isoform- and lipid composition-dependent kinetics of A{beta} fibril formation with clear isoform-dependent lipid effects at a PL ratio of 1:5. Early co-assembly stages of six A{beta}40/A{beta}42 with DMPC and DMPC/CHOL lipids at this PL ratio are then examined by MD. MD results indicate that lipids alter the secondary structure more and interact more avidly with A{beta}40 than with A{beta}42. In turn, A{beta}40 increases the planar order of lipid bicelles more than A{beta}42. Unlike A{beta}40, A{beta}42 decreases the nematic order of CHOL. In DMPC/CHOL lipids, A{beta}42 but not A{beta}40 exhibits two A{beta}-lipid interaction modes associated with mutually exclusive A{beta}42-lipid and A{beta}42-A{beta}42 interactions. Our MD findings provide a plausible explanation for an experimentally observed increase in the lag time for A{beta}40 fibril formation in DMPC lipids and even more so in DMPC/CHOL lipids that contrasts with much smaller effects of lipids on A{beta}42 fibril formation and offer additional atomistic-level insights into A{beta} co-assembly with lipids amenable to experimental testing.

Ferroptosis has attracted significant attention for its potential to treat drug resistant tumors, along with its role in neurodegeneration and ischemia. Several ferroptosis targets were identified in the past decade, yet it is also highly affected by cellular metabolism and epithelial or mesenchymal phenotypes which limits the utility of these targets across diverse cell and tissue types. To address this, we utilized chemo-resistant cancer cell lines with different metastatic potential and tissue origins and discovered a common role of cytoplasmic ion changes during ferroptosis. We observed significant changes in intracellular potassium, sodium and chloride levels upon induction of ferroptosis. Therefore, we screened several ion channel inhibitors and activators (i.e. modulators) and identified Kv7.2 and CFTR modulators influence ferroptosis execution despite the fact that these cell lines do not express these proteins. We demonstrated that clinically approved modulators of CFTR and Kv7.2 can inhibit ferroptosis which indicates that these inhibitors could be repurposed as treatments in ferroptosis-associated diseases such as neurodegeneration and kidney diseases independent of ion channel expression. These modulators also influenced potassium and chloride levels in different contexts indicating off-target effects may also lead to changes in ion levels and may serve as a ferroptosis target. Further, we demonstrated that sustained imbalance of sodium, potassium and chloride ions with ferroptosis inducer salinomycin and digoxin, a Na+/K+ ATPase inhibitor, enhanced ferroptosis. The combination of salinomycin and digoxin successfully enhanced ferroptotic cell death across both cell lines with different metabolic signatures in vitro, offering a novel ferroptosis target. This combination significantly reduced tumor growth in vivo compared with the single agent alone, offering a new therapeutic modality against drug resistant tumors.

The aim of this study was to develop a framework for assessing the incremental value of decisions on drug amount, timing, input structure, cessation and restart. The LOGOS (Longitudinal Optimization of Global Outcomes and Strategies) approach combines matched nested regimen classes, mechanism-specific benchmarks and evidence requirements in a single longitudinal comparison. Thus, the contribution of additional regimen freedom is assessed separately from improvements due to different resources, constraints or evaluation horizons. Nested feasible classes have nondecreasing objective suprema, whereas a strict gain requires a feasible witness above a valid bound for the smaller class. The approach is illustrated through analytical examples of periodic smoothing, matched desensitization, relaxed exposure allocation, finite-course loading and safety after cessation. In a normalized finite Emax model, integrated benefit increased from 0.629606 for three equal boluses to 0.672945 after optimization of amounts, 0.677043 for an amount-and-time candidate and 0.679553 for a globally certified loading-maintenance-coast solution. The adjacent observed gains were 6.88%, 0.61% and 0.37%, respectively, and all schedules satisfied the full-tail injury bound. In addition, a periodic factorial example separated response curvature from a joint recovery and repair process, including a null case and results dependent on feasibility constraints. The LOGOS framework allows assessment of whether additional regimen flexibility improves benefit, provides no added value or restores feasibility. These dimensionless examples support conditional mathematical and model-based conclusions; clinical superiority and safe restart in patients have not been established.

Faster ultrahigh-resolution (UHR) photon-counting CT acquisitions facilitate breath-hold imaging, but identical reconstruction settings may conceal differences in spatial resolution. To assess spatial resolution and depiction of small airways and venous stents across helical UHR acquisition configurations. Stationary wire, airway, and venous-stent phantoms underwent one acquisition per configuration using five configurations on a dual-source scanner at 120 kVp. Reconstructions used 0.2- and 0.4-mm thicknesses, the same sharp kernel, and iterative reconstruction strength 1. We measured the full-width at half-maximum of the slice sensitivity profile (SSP) and spatial frequency at 20% of the modulation transfer function (MTF20). Rank-based F tests with Holm adjustment assessed configuration differences in resolution and variability across slices. Airway detection used a profile contrast-to-noise criterion; stent assessment was qualitative. Calculated acquisition time for 400-mm coverage decreased from 9.80 to 1.30 seconds between the slowest and fastest configurations. At 0.2-mm reconstruction thickness, median SSP increased from 0.316 to 0.496 mm, and median MTF20 decreased from 2.870 to 2.059 lp/mm (both P<.001). Interquartile ranges increased from 0.047 to 0.197 mm for SSP and from 0.256 to 0.654 lp/mm for MTF20 (both variability P<.001). Increasing reconstruction thickness to 0.4 mm increased SSP by a median 0.221 mm across 2459 matched slice-wire measurements (P<.001). At 0.2-mm reconstruction thickness, 0.4- and 0.6-mm lumens with 0.2-mm walls were detected only in the slowest configuration. Faster configurations showed less distinct stent struts and greater adjacent-wall distortion. Faster helical UHR photon-counting CT configurations had poorer spatial resolution despite identical nominal slice thickness. Consistent acquisition and reconstruction settings are important for longitudinal comparisons.

Sepsis mimics resemble sepsis clinically but may require different treatments. We evaluated whether inflammatory protein profiles could distinguish these conditions in a retrospective case-control study using 4 prospective Japanese cohorts. The study included 34 adults with sepsis and 46 with sepsis mimics. We measured 92 proteins using Olink, assessed between-group differences with false discovery rate (FDR) adjustment, and evaluated 3 prediction models using repeated nested cross-validation. No protein met the FDR threshold; the lowest FDR-adjusted P value was 0.345. The primary elastic-net model had an area under the receiver operating characteristic curve of 0.442 (95% confidence interval, 0.315-0.574), based on cross-validation predictions averaged for each participant. Random forest and gradient boosting also showed no clear evidence of discrimination above chance. Confidence intervals included chance performance in both the all-serum and quality-control-Pass sensitivity analyses. In this cohort, a single measurement of an inflammation-focused protein panel showed limited discrimination between sepsis and sepsis mimics. Integration with clinical information and longitudinal measurements warrants further investigation.

Quantitative measurement of fetal hemoglobin (HbF) using high-performance liquid chromatography (HPLC) is standard but expensive and widely unavailable in low-income settings. The newer Gazelle method, which employs single-use alkaline electrophoresis cartridges, provides a portable and relatively inexpensive potential alternative. In the current study, we tested 3055 venous blood samples collected from 1780 children with sickle cell anemia who were enrolled into the H-PRIME trial by both Gazelle and the Bio-Rad D-10 HPLC instrument, before and during hydroxyurea treatment. Data reinterpretation was required to quantify accurately high HbF values derived using the D-10. Mean (standard deviation) baseline HbF values by HPLC and Gazelle (N=2042) were 11.4% (SD 5.9%) versus 16.3% (SD 7.5%), respectively, while mean on-treatment HbF values (N=1013) were 18.9% (SD 9.0%) versus 25.4% (SD 11.5%), respectively. While overall agreement was good (Lins concordance 0.76, 95% CI 0.75-0.78), HbF values derived using Gazelle were modestly higher than HPLC values (Bland-Altman agreement mean +5.4%, 95%CI -2.4% to +13.2%), with the difference between measures increasing at higher HbF percentages. With strong concordance and more easily interpretable HbF values, Gazelle represents an attractive and inexpensive alternative to HPLC for HbF quantification in the low-income settings where most patients with sickle cell anemia live.

Chromosome was "self-assembled" into a complex three-dimensional topology in the nucleus, which provided the structural basis for important biological processes such as gene expression regulation and cell differentiation. The current understanding of the three-dimensional organization of the genome is based on high-throughput sequencing and imaging technologies to visualize the conformation of chromatin in its native environment. This study proposes a new research perspective and technical means to use fusion genes to determine the interaction information and three-dimensional structure of chromosomes. This three-dimensional information provided by the fusion gene spectrum not only shows the universality of the spatial layout of chromosomes, but also increases the interpretability of the occurrence of fusion genes. The chromatin conformation capture data of multiple tissues and periods is significantly correlated with the strength of fusion genes. For example, the frequency of trans-interaction between chromosomes 11 and 17 was the highest, and the frequency of fusion gene occurrence corresponding to KMT2A on chromosome 11 was the highest. Therefore, we infer that this structural universality is a common feature of the human genome, which provides a theoretical basis and data for predicting chromosome fusion events.

Moment-closure methods provide a widely used approach for approximating low-order statistics of stochastic biochemical reaction networks, but the resulting closed equations may exhibit physically meaningless dynamics. Here, we systematically investigate the stability of second-order moment-closure approximations for a bursty gene expression model with nonlinear protein degradation. We consider the Gaussian, log-normal, and negative-binomial closures. By analyzing the resulting dynamical systems, we show that the Gaussian closure fails the proposed stability criterion for all parameter values, whereas both the log-normal and negative-binomial closures possess a unique physically accessible fixed point and are stable if and only if kB(1 + 2B) [&ge;] 1, where B is the mean burst size and k is the dimensionless protein synthesis rate. Moreover, this condition is automatically satisfied when the steady-state mean protein copy number is at least one, suggesting that it is mild in biologically relevant regimes. Numerical comparisons over a broad parameter range further show that the negative-binomial closure is the most accurate, while the Gaussian closure is the least accurate. We further develop higher-order negative-binomial moment-closure schemes and demonstrate that increasing the closure order substantially improves predictive accuracy. These results highlight the importance of considering dynamical stability, in addition to numerical accuracy, when assessing moment-closure approximations.

High-speed atomic force microscopy (HS-AFM) experiments provide unique insights into nanoscale biological processes by directly visualizing the surface topography dynamics of biomolecules, such as proteins, over extended periods of time and generating large imaging data sets. However, the lack of computational methods for automated data analysis has thus far limited the full utilization of these data for the quantitative understanding of single-molecule dynamic. Here, we present the AFM Analysis Arena (A3) within BioAFMviewer as a software platform for automated, high-throughput quantitative analysis of AFM topographic imaging data within a user-friendly interactive graphical interface, implementing a convenient workflow from raw HS-AFM (AFM) imaging to statistical analysis. In several applications to HS-AFM imaging data, we demonstrate methods of automatized molecule detection and dynamic tracking to highlight the available capacities, aiming to transform HS-AFM measurements from observations towards an analytical tool in nanoscale biology.

Nucleolar alterations are characteristic features of cancer cells; however, how changes in the overall morphology relate to internal subcompartment organization and ribosomal RNA (rRNA) dynamics remains unclear. In this study, the nucleolar organization of non-transformed MCF-10A mammary epithelial cells and MDA-MB-231 breast cancer cells was quantitatively compared. MDA-MB-231 cells exhibited differences in nucleolar morphology, accompanied by altered spatial organization of the dense fibrillar component and granular component, and accelerated intranucleolar trafficking of nascent rRNA. Pharmacological perturbation with SU056 shifted multiple structural and dynamic parameters toward an MCF-10A-like profile. Conversely, transient physicochemical perturbation with 1,6-hexanediol induced several MDA-MB-231-like nucleolar features in MCF-10A cells, which returned to their untreated profile after washout. These findings support the view that cancer-associated nucleolar alterations are an integrated reorganization across morphological, spatial, and dynamic dimensions. The observed reversibility further indicates that cancer-associated nucleolar organization can be remodeled in response to perturbations. By linking the morphology, subcompartment organization, and nascent rRNA dynamics within a common framework, this study provides a basis for exploring the establishment, maintenance, and remodeling of distinct nucleolar profiles in cancer.

Alkaline ceramidase 2 (ACER2) catalyzes the hydrolysis of ceramides into sphingosine and free fatty acids, playing crucial roles in regulating various biological processes, including but not limited to cell proliferation, survival, and the DNA damage response. However, a pharmacological tool that regulates its catalytic and signaling functions remains to be developed. In this study, N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]dodecane-1-sulfonamide (KPB82) was identified as a potent and selective ACER2 inhibitor. In vitro enzymatic assays showed that KPB82 inhibited ACER2 with an IC50 of 190 nM, while displaying minimal activity against acid and neutral ceramidases (IC50> 100 {micro}M) and no significant inhibition of alkaline ceramidase 1 or alkaline ceramidase 3. Enzymatic kinetic studies revealed that KPB82 is a competitive inhibitor of ACER2. The three-dimensional structure of ACER2 was modeled in the AlphaFold3 server, revealing key Zn2+-coordinating histidine residues and a substrate-binding pocket. Molecular docking studies demonstrated that KPB82 binds ACER2 within this pocket, forming hydrogen bonds with the catalytically important amino residues, Asp93 and His215, and interacting with several hydrophobic residues. Lactate dehydrogenase release assays revealed that KPB82 significantly reduced cytotoxicity in HeLa cells overexpressing ACER2, as well as in HCT116 cells, RT4 cells, and human umbilical vein endothelial cells treated with the DNA-damaging agent doxorubicin. Lipidomic analysis showed that KPB82 inhibited sphingosine production in response to ACER2 overexpression or doxorubicin treatment. Collectively, these findings demonstrate that KPB82 is a potent and selective ACER2 inhibitor capable of suppressing ACER2-dependent biological processes and highlight its potential as a tool for studying ceramide metabolism and ACER2-mediated signaling pathways.

Abstract Background. Tuberous Sclerosis Complex (TSC) is caused by inactivating mutations in either the TSC1 or TSC2 genes, leading to activation of the mammalian target of rapamycin complex 1 (mTORC1) and unhindered cell growth and proliferation. The epithelium of TSC renal cysts in both mice and humans is composed of proliferating A-intercalated (A-IC) cells. The exact molecular mechanism of kidney cystogenesis in TSC remains speculative. Hypothesis.Superfluous cell proliferation driven by mTORC1 activation increases metabolic demand, causing oxidative stress and excess reactive oxygen species. If unchecked, this overwhelms antioxidant defenses and causes cell death. In TSC kidney cystogenesis, Nuclear Factor Erythroid 2-Related Factor 2 (NFE2L2), also known as NRF2, serves as the "master regulator" of antioxidant and anti-inflammatory responses, enabling cells to survive and proliferate by clearing the toxic environment and supplying nutrients and fuel. Results. RNA-seq and proteomics, along with western blot analysis, showed robust downregulation of Fumarate Hydrase 1 (FH1) and upregulation of NRF2, STAT3, and HIF1 in kidneys from TSC mice with moderate or heavy cyst burden. Confocal microscopy and immunohistochemical staining on kidney sections, and/or western blot studies on nuclear and cytoplasmic fractions, showed nuclear localization of NRF2, STAT3, and HIF1. In cyst-lining cells in TSC mouse models, FH1 downregulation was associated with inactivating succination of KEAP1 in immunoprecipitation experiments, promoting NRF2 nuclear localization in A-IC cells lining the cysts. NRF2 nuclear localization was associated with ectopic induction of the glutamine transporter SLC38A3 (SNAT3) on the basolateral membrane and activation of the NH3/NH4+ transporters RHCG and RHBG in A-IC cells lining the cysts. Twenty-four h urine NH3/NH4+ excretion rates increased significantly in Tsc1 KO vs. WT mice. The activation of NRF2, STAT3, and HIF1 can drive metabolic reprogramming and activate survival genes in proliferating cells. Together with SLC38A3 induction and upregulation of other glutamine and NH3/NH4+ transporters, these factors activate glutaminolysis and aerobic glycolysis, supplying nutrients to proliferating cystic epithelial cells and supporting cyst expansion in TSC. Consistent with this central role for glutaminolysis in kidney cystic epithelium and TSC cystogenesis, we find a significant reduction in kidney cyst burden in Tsc1 KO mice on a glutamine-free diet. Conclusions. NRF2 plays a critical role in antioxidant defense. Along with STAT3 and HIF1, NRF2 is a key player in metabolic reprogramming through glutaminolysis, which supplies nutrients to proliferating cystic epithelial cells and supports cyst expansion in TSC. These findings suggest that inhibiting or inactivating NRF2, alone or in combination with HIF1 or STAT3, may represent a potential treatment strategy for kidney lesions in TSC.

Whether the homologous parallel triplex (HP-triplex, formed by dsDNA and a homologous ssDNA) acts as an intermediate prior to DNA recombination remains controversy, owing to the intrinsic instability and lake of experimental data. Here, we report a HP-triplex that is stable at least at 35 oC under protein-free conditions. Two complementary circular ssDNAs were first hybridized at a 1:1 ratio to form an LR-chimera containing both Z-DNA and B-DNA. Then the circular homologous ssDNA (T-rich) was found to easily hybridize with this LR-chimera to form the HP-triplex (strand ratio of 2:1), involving both left- and right-handed triplex parts under the topological constraint. The third strand locates in the major groove and forms hydrogen bonds with both bases in each base pair of dsDNA without obvious strand exchange. Our data indicate that the third-strand ssDNA may first bind to the B-DNA region, followed by association with the Z-DNA region to assemble the full triplex structure. Structural simulations and analyses reveal that topological constraint imposed by the circular architecture raises the energy barrier for strand displacement, thereby stabilizing this triplex. In summary, the dynamic HP triplex can be stabilized via a Z B junction structure that mimics the in vivo one formed by negative supercoiling. These findings demonstrate that HP-triplex may act as an intermediate for DNA recombination, anti-gene strategy, and other interactions between dsDNA and homologous ssDNA or RNA.