Native Characterization of Noncanonical Nucleic Acid Thermodynamics via Programmable Dynamic DNA Chemistry.

Folding thermodynamics, quantitatively described using parameters such as ΔGfold°, ΔHfold°, and ΔSfold°, is essential for characterizing the stability and functionality of noncanonical nucleic acid structures but remains difficult to measure at the molecular level. Leveraging the programmability of dynamic deoxyribonucleic acid (DNA) chemistry, we introduce a DNA-based molecular tool capable of performing a free energy shift assay (FESA) that directly characterizes the thermodynamics of noncanonical DNA structures in their native environments. FESA operates by the rational design of a reference DNA probe that is energetically equivalent to a target noncanonical nucleic acid structure in a series of toehold-exchange reactions, yet is structurally incapable of folding. As a result, a free energy shift (ΔΔGrxn°) is observed when plotting the reaction yield against the free energy of each toehold-exchange. We mathematically demonstrated that ΔGfold°, ΔHfold°, and ΔSfold° of the analyte can be calculated based on ΔΔGrxn°. After validating FESA using six DNA hairpins by comparing the measured ΔGfold°, ΔHfold°, and ΔSfold° values against predictions made by NUPACK software, we adapted FESA to characterize noncanonical nucleic acid structures, encompassing DNA triplexes, G-quadruplexes, and aptamers. This adaptation enabled the successful characterization of the folding thermodynamics for these complex structures under various experimental conditions. The successful development of FESA marks a paradigm shift and a technical advancement in characterizing the thermodynamics of noncanonical DNA structures through molecular tools. It also opens new avenues for probing fundamental chemical and biophysical questions through the lens of molecular engineering and dynamic DNA chemistry.

Coding Intrinsic Disorder into DNA Hybridization Probes Enables Discrimination of Single Nucleotide Variants over Wide and Tunable Temperature Ranges.

DNA hybridization probes are commonly used tools to discriminate clinically important single nucleotide variants (SNVs) but often work at elevated temperatures with very narrow temperature intervals (ΔT). Herein, we investigated the thermodynamic basis of the narrow ΔT both in silico and experimentally. Our study revealed that the high entropy penalty of classic hybridization probe designs was the key attributor for the narrow ΔT. Guided by this finding, we further introduced an entropy-compensate probe (Sprobe) design by coding intrinsic disorder into a stem-loop hybridization probe. Sprobe expanded ΔT from less than 10 °C to over 30 °C. Moreover, both ΔT and the optimal reaction temperature can be fine-tuned by simply altering the length of the loop domain. Sprobe was clinically validated by analyzing EGFR L858R mutation in 36 pairs of clinical tumor tissue samples collected from lung cancer patients, which revealed 100 % clinical sensitivity and specificity. We anticipate that our study will serve as a general guide for designing thermal robust hybridization probes for clinical diagnostics.

Sex hormone influence on female-biased autoimmune diseases hints at puberty as an important factor in pathogenesis.

The majority of autoimmune diseases affect more women than men, suggesting an important role for sex hormones in regulating immune response. Current research supports this idea, highlighting the importance of sex hormones in both immune and metabolic regulation. Puberty is characterized by drastic changes in sex hormone levels and metabolism. These pubertal changes may be what forms the gulf between men and women in sex bias towards autoimmunity. In this review, a current perspective on pubertal immunometabolic changes and their impact on the pathogenesis of a select group of autoimmune diseases is presented. SLE, RA, JIA, SS, and ATD were focused on in this review for their notable sex bias and prevalence. Due to both the scarcity of pubertal autoimmune data and the differences in mechanism or age-of-onset in juvenile analogues often beginning prior to pubertal changes, data on the connection between the specific adult autoimmune diseases and puberty often relies on sex hormone influence in pathogenesis and established sex differences in immunity that begin during puberty.

Toehold-Exchange-Based Activation of Aptamer Switches Enables High Thermal Robustness and Programmability.

Aptamer switches are attractive nature-inspired tools for developing smart materials and nanodevices. However, the thermal robustness and programmability of current aptamer switches are often limited by their activation processes that are coupled with high reaction enthalpy. Here, we present an enthalpy-independent activation approach that harnesses toehold-exchange as a general framework to design aptamer switches. We demonstrate mathematically and experimentally that this approach is highly effective in improving thermal robustness and thus leads to better analytical performances of aptamer switches. Enhanced programmability is also demonstrated through fine-grained and dynamic tuning of effective affinities and dynamic ranges, as well as the construction of a synthetic DNA network that resembled biological signaling cascades. Our study not only enriches the current toolbox for engineering and controlling synthetic molecular switches but also offers new insights into their thermodynamic basis, which is critical for diverse synthetic biological designs and applications.

G4LDB 2.2: a database for discovering and studying G-quadruplex and i-Motif ligands.

Noncanonical nucleic acid structures, such as G-quadruplex (G4) and i-Motif (iM), have attracted increasing research interests because of their unique structural and binding properties, as well as their important biological activities. To date, thousands of small molecules that bind to varying G4/iM structures have been designed, synthesized and tested for diverse chemical and biological uses. Because of the huge potential and increasing research interests on G4-targeting ligands, we launched the first G4 ligand database G4LDB in 2013. Here, we report a new version, termed G4LDB 2.2 (http://www.g4ldb.com), with upgrades in both content and function. Currently, G4LDB2.2 contains >3200 G4/iM ligands, ∼28 500 activity entries and 79 G4-ligand docking models. In addition to G4 ligand library, we have also added a brand new iM ligand library to G4LDB 2.2, providing a comprehensive view of quadruplex nucleic acids. To further enhance user experience, we have also redesigned the user interface and optimized the database structure and retrieval mechanism. With these improvements, we anticipate that G4LDB 2.2 will serve as a comprehensive resource and useful research toolkit for researchers across wide scientific communities and accelerate discovering and validating better binders and drug candidates.

The CRISPR-Cas toolbox for analytical and diagnostic assay development.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems have revolutionized biological and biomedical sciences in many ways. The last few years have also seen tremendous interest in deploying the CRISPR-Cas toolbox for analytical and diagnostic assay development because CRISPR-Cas is one of the most powerful classes of molecular machineries for the recognition and manipulation of nucleic acids. In the short period of development, many CRISPR-enabled assays have already established critical roles in clinical diagnostics, biosensing, and bioimaging. We describe in this review the recent advances and design principles of CRISPR mediated analytical tools with an emphasis on the functional roles of CRISPR-Cas machineries as highly efficient binders and molecular scissors. We highlight the diverse engineering approaches for molecularly modifying CRISPR-Cas machineries and for devising better readout platforms. We discuss the potential roles of these new approaches and platforms in enhancing assay sensitivity, specificity, multiplexity, and clinical outcomes. By illustrating the biochemical and analytical processes, we hope this review will help guide the best use of the CRISPR-Cas toolbox in detecting, quantifying and imaging biologically and clinically important molecules and inspire new ideas, technological advances and engineering strategies for addressing real-world challenges such as the on-going COVID-19 pandemic.

The Construction of DNA Logic Gates Restricted to Certain Live Cells Based on the Structure Programmability and Aptamer-Cell Affinity of G-Quadruplexes.

DNA computation is considered a fascinating alternative to silicon-based computers; it has evoked substantial attention and made rapid advances. Besides realizing versatile functions, implementing spatiotemporal control of logic operations, especially at the cellular level, is also of great significance to the development of DNA computation. However, developing simple and efficient methods to restrict DNA logic gates performing in live cells is still a challenge. In this work, a series of DNA logic gates was designed by taking full advantage of the diversity and programmability of the G-quadruplex (G4) structure. More importantly, by further using the high affinity and specific endocytosis of cells to aptamer G4, an INHIBIT logic gate has been realized whose operational site is precisely restricted to specific live cells. The design strategy might have great potential in the field of molecular computation and smart bio-applications.

Probing the Formation Kinetics and Thermodynamics with Rationally Designed Analytical Tools Enables One-Pot Synthesis and Purification of a Tetrahedral DNA Nanostructure.

The development of robust analytical tools capable of probing the formation kinetics and thermodynamics of DNA nanostructures is a crucial step toward better understanding and manufacturing of diverse DNA-based materials. Herein, we introduce a real-time fluorescence anisotropy assay and rationally designed DNA reaction termination probes (DRTPs) as a set of new tools for exploring the formation mechanisms of DNA nanostructures. We deployed these tools for probing the formation of a classic tetrahedral DNA nanostructure (TDN) as a model system. Our tools revealed that the formation of TDN was dominated by simultaneous hybridization, whereas its undesired side products were caused mainly through step-wise hybridization. An optimal reaction temperature exists that favors the formation of TDN over side products. With insight into the TDN formation mechanism, we further engineered magnetic DRTPs to achieve single-step purification of TDN, enabling 10-fold improvement in the ratio between the targeted TDN and undesired side products without tedious procedures or bulky instruments. Combining the optimal reaction and purification conditions, we finally demonstrated the one-pot synthesis and purification of TDN. The analytical techniques offered in this work may hold potential to find wide applications and inspire new analytical methods for structural DNA nanotechnology.

Information processing using an integrated DNA reaction network.

Living organisms use interconnected chemical reaction networks (CRNs) to exchange information with the surrounding environment and respond to diverse external stimuli. Inspired by nature, numerous artificial CRNs with a complex information processing function have been recently introduced, with DNA as one of the most attractive engineering materials. Although much progress has been made in DNA-based CRNs in terms of controllable reaction dynamics and molecular computation, the effective integration of signal translation with information processing in a single CRN remains to be difficult. In this work, we introduced a stimuli-responsive DNA reaction network capable of integrated information translation and processing in a stepwise manner. This network is designed to integrate sensing, translation, and decision-making operations by independent modules, in which various logic units capable of performing different functions were realized, including information identification (YES and OR gates), integration (AND and AND-AND gates), integration-filtration (AND-AND-NOT gate), comparison (Comparator), and map-to-map analysis (Feynman gate). Benefitting from the modular and programmable design, continuous and parallel processing operations are also possible. With the innovative functions, we show that the DNA network is a highly useful addition to the current DNA-based CRNs by offering a bottom-up strategy to design devices capable of cascaded information processing with high efficiency.

Recent advances in nanomaterial-enhanced enzyme-linked immunosorbent assays.

Despite serving as a gold standard for protein analysis, the classic enzyme-linked immunosorbent assay (ELISA) is currently challenged by the ever-increasing needs of sensitivity and simplicity. Towards the ongoing needs, recent advances in nanomaterials have offered numerous promising tools for enhancing the performance and broadening the applicability of ELISA. In this review, we highlight nanomaterial-enabled strategies that drastically improve the assay performance without significantly altering the classic ELISA format. Particular attention will be focused on the functional roles of nanomaterials as novel readout systems in ELISA, including those serving as substrate-alternatives, enzyme-alternatives, and non-enzymatic signal amplifiers.

A supramolecular aggregation-based constitutional dynamic network for information processing.

Concepts and strategies offered by constitutional dynamic chemistry (CDC) hold great promise for designing molecular computing systems adaptive to external environments. Despite demonstrable success in storing and processing chemical information using CDC, further employment of such constitutional dynamic networks (CDNs) for processing more complex digital information has not been realized yet. Herein, we introduced a supramolecular CDN based on the aggregation of cyanine MTC (Agg-CDN), which is composed of four reversibly interconvertible constituents, i.e. monomers, dimers, J-aggregates, and H-aggregates. We demonstrated that the equilibrated Agg-CDN is reconfigurable through constituent exchange in response to well-defined chemical inputs. More importantly, the equilibrated states of the Agg-CDN are spectroscopically distinguishable because of the unique optical properties of MTC. We further tuned the Agg-CDN to at least nine unique states for transforming the chemical inputs into digital outputs, and successfully employed it for encoding and encrypting complex digital information, such as multi-pixel images.

SIP1 serves a role in HBx­induced liver cancer growth and metastasis.

Hepatitis B virus (HBV) has been revealed to be involved in the development of hepatocellular carcinoma. However, the mechanism remains to be fully elucidated. Smad­interacting protein 1 (SIP1) is a transcriptional repressor, which serves a pivotal role in cell metastasis. In the present study, the role of SIP1 in HBx­induced hepatocyte EMT and cancer aggressiveness was examined. It was found that HBV X protein (HBx) increased the expression of SIP1 and recruited it to the promoter of E­cadherin, resulting in depression of the transcription of E­cadherin. Histone deacetylase 1 was also found to be involved in the repressive complex formation. Furthermore, in an orthotopic tumor transplantation model in vivo, HBx promoted tumor growth and metastasis, whereas the knockdown of SIP1 attenuated the effect of HBx. These results indicate a novel mechanism for the development of HBV­related liver cancer.

Construction of a novel DNA-based comparator and its application in intelligent analysis.

In this work, a novel and general comparator was constructed based on cascaded strand displacement reactions and DNA hybridization and its potential in intelligently weighing the quantitative predominance of two targets was explored in a complex biological matrix, which not only enriches the information processing mode of DNA computation but also provides an instructive way to deal with quantitative analyzing tasks in further DNA-based logic sensors.

A resettable supramolecular platform for constructing scalable encoders.

A supramolecular platform prototype for implementing resettable encoding functions was designed, which could be configured into a series of encoders, from 4-to-2 to 7-to-3, and even 14-to-4 ECs.

Versatile and Homogeneous DNA Tetraplex Platform for Constructing Label-Free Logic Devices: From Design to Application.

The design of DNA-based logic circuits has become an active research field in DNA nanotechnology and holds great potential in intelligent bioanalysis. To date, although many DNA-based logic systems have been realized, the implementation of advanced logic functions is still challenging, especially with simple and homogeneous compositions. Herein, by integrating two DNA tetraplex structures (G-quadruplex and i-motif), a completely label-free logic platform with high scalability was established, with which a series of advanced functions were realized, including arithmetic (adders and subtractors) and nonarithmetic ones (majority and dual-transfer gates). Furthermore, the platform was also applied as an intelligent biosensor to coanalyze two cancer-related micro-RNAs with high sensitivities and specificities. Considering the excellent versatility, expandability, and biocompatibility, the platform may promote the development of DNA computing and hold great potential in multiparameter sensing and medical diagnosis.

A Visibly Observable, Programmable Supramolecular Logic Platform and Its Application in Smart Thiols Sensing.

Molecular computation is increasingly attractive as a tool for medical and biological research because of its programmability and controllability. Herein, a novel visibly observable supramolecular system that can execute multi-level logic functions on a uniform platform was constructed. By employing some programming factors, we succeeded in not only constructing a whole set of contrary logic pairs, but also building up a logic network that can implement advanced functions. Further, the platform is applied to sense thiols in specific environments. The developed method can efficiently filter signals of thiols in intracellular conditions and measure cysteine levels quantitatively in serum conditions. The visual readout makes the method particularly suitable for point-of-care testing. The supramolecule-based platform illustrates not only an incremental advance for the construction of programmable molecular logic systems, but also viable applications in intelligent thiol analysis.

Recent Progress in Fluorescence Signal Design for DNA-Based Logic Circuits.

DNA-based logic circuits, encoding algorithms in DNA and processing information, are pushing the frontiers of molecular computers forward, owing to DNA's advantages of stability, accessibility, manipulability, and especially inherent biological significance and potential medical application. In recent years, numerous logic functions, from arithmetic to nonarithmetic, have been realized based on DNA. However, DNA can barely provide a detectable signal by itself, so that the DNA-based circuits depend on extrinsic signal actuators. The signal strategy of carrying out a response is becoming one of the design focuses in DNA-based logic circuit construction. Although work on sequence and structure design for DNA-based circuits has been well reviewed, the strategy on signal production lacks comprehensive summary. In this review, we focused on the latest designs of fluorescent output for DNA-based logic circuits. Several basic strategies are summarized and a few designs for developing multi-output systems are provided. Finally, some current difficulties and possible opportunities were also discussed.

Room temperature oxidative desulfurization with MoO3 subnanoclusters supported on MCM-41.

Subnano MoO3/MCM-41 was successfully prepared through doping (NH4)6Mo7O24 in the synthesis process of MCM-41. The morphology of MoO3/MCM-41 was visually observed by TEM and HADDF-STEM. N2 sorption, XPS and Raman were further applied to investigate the structure of the material. MoO3/MCM-41 was used in the oxidative desulfurization process with tert-butyl hydroperoxide as oxidant. MoO3/MCM-41 showed outstanding catalytic activity and recycling ability at room temperature.

Identification of serum ß-catenin as a biomarker in patients with HBV-related liver diseases.

BACKGROUND: Substantial evidence indicates that ß-catenin is a pivotal regulator that contributes to the initiation and development of various types of diseases. Recently, ß-catenin can be detected in human serum and also reported to be correlated with several disease progression in a little research. However, very little is known about the relationship between serum ß-catenin and HBV-related liver disease. METHODS: Serum levels of ß-catenin, from 77 patients with chronic hepatitis B (CHB), 63 patients with hepatitis B associated liver cirrhosis (HBLC), 61 patients with hepatocellular carcinoma (HCC), 41 healthy HBV carriers (HHCs) and 78 healthy controls (HCs) were measured by ELISA. Correlations of serum ß-catenin with viral replication and liver necroinflammation parameters were analyzed. The receiver operating characteristic (ROC) curve was used to assess the discriminating power of serum ß-catenin to grade different stages of HBV-related disorders. Human hepatic cell line L02 was transfected with a HBV plasmid, and ß-catenin levels and the underlying mechanism were analyzed. RESULTS: Chronic hepatitis B and HBLC patients but not HHC or HCC showed significantly higher serum ß-catenin levels than HCs. ß-catenin levels were not correlated with HBV DNA levels but were correlated with necroinflammation parameters. HBV-infected cell model showed elevated levels of phosphorylation at Ser473 in Akt (p-Akt), phosphorylation at Ser9 in GSK3ß (p-GSK3ß) and ß-catenin, all of which was blocked by treatment with Akt inhibitor LY294002. Additionally, ROC analysis of ß-catenin for discriminating patients with CHB from HHCs, which yielded an AUC of 0.71 (cutoff value, 42 pg/mL; 95% CI 0.61-0.81) with 64.93% sensitivity, 73.17% specificity and 69.05% accuracy. ROC analysis of ß-catenin for discriminating patients with HCC from chronic HBV infection mainly including CHB and HBLC, which yielded an AUC of 0.75 (cutoff value, 42 pg/mL; 95% CI 0.67-0.83) with 66.43% sensitivity, 75.41% specificity and 70.92% accuracy. CONCLUSIONS: HBV infection enhances ß-catenin expression by activating Akt/GSK3ß signaling. Serum ß-catenin levels are correlated with necroinflammation parameters but not with viral load. Serum ß-catenin has potential to discriminate the phase of HBV-related disorders, particularly predicts the patients with CHB from HHCs and also predicting HCC form chronic HBV infection.

Intelligent Sensors of Lead Based on a Reconfigurable DNA-Supramolecule Logic Platform.

The lead (Pb) hazard is not only in connection with the concentration of Pb2+ but also closely related to the ambience which affects its mobility and the synergistic toxicity with other ions. However, most of the existent methods focus highly on detecting Pb2+ concentration accurately but can seldom reflect the pollution-related information in actual samples, thereby limiting their pragmatic applications. In this work, a DNA-supramolecule logic platform was established, which can be configurated to implement three information process functions and act as three unique intelligent sensors of Pb. The demultiplexer that can split signal flow was used to determine Pb2+ in different pH conditions; the multiplexer that can alternate signal channels was applied to detect Pb2+ or Ag+ selectively; and the decoder that can extract information was utilized to test Pb2+ and the coexisted Ni2+ simultaneously. All three intelligent sensors based on the logic prototypes present practicable sensitivities and specificities. Considering its flexibility, scalability, and reconfigurability, we believe the logic platform may provide new solutions to process sophisticated information and implement intelligent analysis in environmental monitoring, biochemical detecting, and medical diagnosis.