Fabrication of circular assemblies with DNA tetrahedrons: from static structures to a dynamic rotary motor.

DNA tetrahedron as the simplest 3D DNA nanostructure has been applied widely in biomedicine and biosensing. Herein, we design and fabricate a series of circular assemblies of DNA tetrahedron with high purity and decent yields. These circular nanostructures are confirmed by endonuclease digestion, gel electrophoresis and atomic force microscopy. Inspired by rotary protein motor, we demonstrate these circular architectures can serve as a stator for a rotary DNA motor to achieve the circular rotation. The DNA motor can rotate on the stators for several cycles, and the locomotion of the motor is monitored by the real-time fluorescent measurements.

Versatile Types of DNA-Based Nanobiosensors for Specific Detection of Cancer Biomarker FEN1 in Living Cells and Cell-Free Systems.

Flap structure-specific endonuclease 1 (FEN1) is overexpressed in various types of human cancer cells and has been recognized as a promising biomarker for cancer diagnosis in the recent years. In order to specifically detect the abundance and activity of this cancer-overexpressed enzyme, different types of DNA-based nanodevices were created during our investigations. It is shown in our studies that these newly designed biosensors are highly sensitive and specific for FEN1 in living cells as well as in cell-free systems. It is expected that these nanoprobes could be useful for monitoring FEN1 activity in human cancer cells, and also for cell-based screening of FEN1 inhibitors as new anticancer drugs.

Sulfinate Based Selective Labeling of 5-Hydroxymethylcytosine: Application to Biotin Pull Down Assay.

We developed an enzyme-free, chemical method to selectively label the epigenetic base, 5-hydroxymethylcytosine (hmC) with versatile sulfinate reagents in aqueous solvent under mild reaction conditions. This method allows efficient single step conjugation of biotin to hmC site in DNA for enrichment and pull down assays.

G-quadruplex Nanowires To Direct the Efficiency and Selectivity of Electrocatalytic CO2 Reduction.

DNA as a medium for electron transfer has been widely used in photolytic processes but is seldom applied to dark reaction of CO2 reduction. A G-quadruplex nanowire (tsGQwire) assembled by guanine tetranucleotides was used to host several metal complexes and further to mediate electron transfer processes in the electrochemical reduction of CO2 catalyzed by these complexes. The tsGQwire modified electrode increased the Faradaic efficiency of cobalt(II) phthalocyanine (CoII Pc) 2.5-folds for CO production than bare CoII Pc electrode, with a total current density of 11.5 mA cm-2 . Comparable Faradaic efficiency of HCOOH production was achieved on tsGQwire electrode when the catalytic center was switched to a GQ targeting Ru complex. The high efficiency and selectivity of electrocatalytic CO2 reduction was attributed to the unique binding of metal complexes on G-quadruplex and electron transfer mediated by GQ nanowire to achieve efficient redox cycling of catalytic centers on the electrode.

Ultrathin Two-Dimensional Covalent Organic Framework Nanosheets: Preparation and Application in Highly Sensitive and Selective DNA Detection.

The ability to prepare ultrathin two-dimensional (2D) covalent organic framework (COF) nanosheets (NSs) in high yield is of great importance for the further exploration of their unique properties and potential applications. Herein, by elaborately designing and choosing two flexible molecules with C3v molecular symmetry as building units, a novel imine-linked COF, namely, TPA-COF, with a hexagonal layered structure and sheet-like morphology, is synthesized. Since the flexible building units are integrated into the COF skeletons, the interlayer stacking becomes weak, resulting in the easy exfoliation of TPA-COF into ultrathin 2D NSs. Impressively, for the first time, the detailed structural information, i.e., the pore channels and individual building units in the NSs, is clearly visualized by using the recently developed low-dose imaging technique of transmission electron microscopy (TEM). As a proof-of-concept application, the obtained ultrathin COF NSs are used as a novel fluorescence sensing platform for the highly sensitive and selective detection of DNA.

Efficient Long-Range Hole Transport Through G-Quadruplexes.

DNA offers a means of long-range charge transport for biology and electric nanodevices. Here, a series of tetra-stranded G-quadruplexes were assembled within a dendritic DNA architecture to explore oxidative charge transport (hole transport) through the G-quadruplex. Efficient charge transport was achieved over 28 Šupon UV irradiation. Over a longer G-quadruplex bridge, hole transport was escalated to a higher efficiency, which resulted in a higher yield than that of the optimal duplex DNA for charge transport, that is, the adenine tract. Efficient long-range hole transport suggests tetra-stranded G-quadruplexes, instead of an oxidation hotspot, hold better potential as an electron conduit than duplex DNA.

Topoisomerase-Based Preparation and AFM Imaging of Multi-Interlocked Circular DNA.

Multi-interlocked circular DNA structures have been in high demand for fabricating complicated functional DNA architectures and nanodevices such as molecular switches, shuttles, and motors. Even though various innovative methods have been developed in the past, creation of multi-interlocked circular DNA structures with defined numbers of DNA molecules and linking patterns is still a challenging task nowadays. Here, we propose a top-down decatenation of kinetoplast DNA as a new approach for creating multi-interlocked circular DNA structures. Through optimizing the amount and reaction time of topoisomerase II, we synthesized completely mutually interlocked tricircular, tetra-circular, and oligo-circular DNA structures, which have not yet been acquirable through any other existing synthetic means. The catenation structures of multiple circular DNA were further verified through atomic force microscopic analysis of the backbone overlapping patterns and the circumference. It accordingly is our expectation that the top-down enzymatic approaches could offer a highly interlocked network with defined numbers of circular DNA with simple protocols, and could consequently be beneficial to the design and fabrication of sophisticated functional molecules and nanodevices in the areas of supramolecular chemistry, DNA nanotechnology, and material science.

Magnetic Fields Facilitate DNA-Mediated Charge Transport.

Exaggerated radical-induced DNA damage under magnetic fields is of great concern to medical biosafety and biomolecular electronic devices. In this report, the effects of an external magnetic field (MF) on DNA electronic conductivity were investigated by studying the efficiencies of photoinduced DNA-mediated charge transport (CT) via guanine damage. Under a static MF of 300 mT, positive enhancements in the decomposition of 8-cyclopropyldeoxyguanosine ((8CP)G) were observed at both the proximal and distal guanine doublets, indicating a more efficient propagation of radical cations and higher electronic conductivity of duplex DNA. MF-assisted CT has shown sensitivity to magnetic field strength, duplex structures, and the integrity of base pair stacking. Spin evolution of charge injection and the alignment of base pairs to the CT-active conformation during radical propagation were proposed to be the two major factors that MF contributes to facilitate DNA-mediated CT. Herein, MF-assisted CT may offer a new avenue for designing DNA-based electronic devices and unraveling MF effects on redox and radical relevant biological processes.

Stability and kinetics of c-MYC promoter G-quadruplexes studied by single-molecule manipulation.

A DNA G-quadruplex (G4) formed at the oncogene c-MYC promoter region functions as a gene silencer. Due to its high stability at physiological K(+) concentrations, its thermodynamics and kinetic properties have not been characterized in physiological solution conditions. In this work, we investigated the unfolding and folding transitions of single c-MYC G4 and several of its truncated or point mutants at 100 mM KCl concentration under mechanical force. We found that the wild type could fold into multiple species, and the major specie has a slow unfolding rate of (1.4 ± 1.0) × 10(-6) s(-1). The force-dependent thermodynamics and kinetic properties of the major specie were obtained by studying a truncated mutant, Myc2345, that contains the G-tracts 2, 3, 4, and 5. As the c-MYC G4 is a prototype of many other intermolecular parallel-stranded G4's, our results provide important insights into the stability of a broad class of promoter G4's which also play a role in transcription regulation and are potential anticancer targets.

5-Hydroxymethylcytosine and 5-formylcytosine containing deoxyoligonucleotides: facile syntheses and melting temperature studies.

An oxidation-based synthetic approach was developed for facile preparation of 5-formyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine phosphoramidites. Upon introducing organic solvent components and copper catalysts, C5-methyl groups of 5-methyl-2'-deoxycytidine and thymidine were readily oxidized to formyl and hydroxyl functionality, respectively. Standard solid phase DNA synthesis and conventional deprotection methods were applicable to synthesize 5-formyl- or 5-hydroxymethyl-cytosine containing DNA oligonucleotides, which were used to study the effect of epigenetic modifications on DNA thermal dynamic stability.

Regulation of telomeric i-motif stability by 5-methylcytosine and 5-hydroxymethylcytosine modification.

The two important epigenetic markers in the human genome, 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC), are involved in gene regulation processes. As a major epigenetic target, cytosines in a C-rich DNA sequence were substituted with mC and hmC to investigate the thermal stability and pH sensitivity of the corresponding i-motifs. Circular Dichroism (CD) studies indicate the formation of i-motifs at acidic pH (<6.5) for mC- and hmC-modified DNA sequences. Thermal denaturation results suggest that DNA i-motifs are stabilized when modified with one or two mCs. However, hypermethylation with mC and single modification with hmC cause destabilization of the structure. A biomimetic crowding agent does not alter the stability effect trends resulting from mC and hmC modifications, though the corresponding i-motifs show elevated melting temperatures without significant changes in pKa values.

8-cyclopropyl-2'-deoxyguanosine: a hole trap for DNA-mediated charge transport.

DNA duplexes containing 8-cyclopropyl-2'-deoxyguanosine ((8CP) G) were synthesized to investigate the effect of the C8-modified deoxyguanosine as a kinetic trap for transient hole occupancy on guanines during DNA-mediated hole transport (HT). Thermal denaturation and CD spectra show that DNA duplexes containing (8CP) G are able to form stable B-form duplexes. Photoirradiation of terminal tethered anthraquinone can induce oxidative decomposition of (8CP) G through DNA HT along adenine tracts with lengths of up to 4.8 nm. Shallow and periodic distance dependence was observed in a long adenine tract with intervening guanines. The efficient charge transport indicates that (8CP) G can electronically couple well with a DNA bridge and form HT-active conformational domains to facilitate transient hole delocalization over an adenine tract.

Neutrophils in cancer: dual roles through intercellular interactions.

Neutrophils, the most abundant immune cells in human blood, play crucial and diverse roles in tumor development. In the tumor microenvironment (TME), cancer cells regulate the recruitment and behaviors of neutrophils, transforming some of them into a pro-tumor phenotype. Pro-tumor neutrophils interact with cancer cells in various ways to promote cancer initiation, growth, and metastasis, while anti-tumor neutrophils interact with cancer cells to induce senescence and death. Neutrophils can also interact with other cells in TME, including T cells, macrophages, stromal cells, etc. to exert anti- or pro-tumor functions. In this review, we will analyze the anti- and pro-tumor intercellular interactions mediated by neutrophils, with a focus on generalizing the mechanisms underlying the interaction of neutrophils with tumor cells and T cells. Furthermore, we will provide an overview of cancer treatment strategies targeting neutrophil-mediated cellular interactions.

Dual-functional DNA nanogels for anticancer drug delivery.

DNA hydrogels with unique sequence programmability on nucleic acid framework manifest remarkable attributes, such as high payload capacities, biocompatibility and biosafety. The availability of DNA nanogels with multimodal functionalities remains limited due to the absence of facile gelation methods applicable at the nanometer scale. Here, we developed a one-step assembly of DNA dendrimers into nanogels (DNG) with couple hundred nanometers size. DNG showed robust stability against physical forces and biological degradation for easy purification and sustainable drug release. Long-term stability either in powder or aqueous solution endows DNG easy for shipping, handling and storage. By encoding dual functionalities into separate branches on DNA dendrimers, DNG can accommodate chemodrugs and aptamers with distinctive loading moduli. DNG significantly enhanced the drug efficacy against cancerous cells while minimizing cytotoxicity towards somatic cells, as demonstrated in vitro and in xenografted mice models of breast cancer. Thus, due to their facile assembly and storage, bi-entity encoding, and inherent biocompatibility, DNG exhibits immense prospects as nanoscale vesicles for the synergistic delivery of multimodal theranostics in anticancer treatments. STATEMENT OF SIGNIFICANCE: DNA nanogels were self-assembled via a facile protocol utilizing a DNA dendrimer structure. These nanogels displayed robust stability against physical forces, permitting long term storage in concentrated solutions or as a powder. Furthermore, they exhibited resilience to biological degradation, facilitating sustained drug release. The bi-entity encoded dendritic branches conferred dual functionalities, enabling both chemodrug encapsulation and the presentation of aptamers as targeting motifs. In vivo investigations confirmed the nanogels provide high efficacy in tumor targeting and chemotherapy with enhanced drug efficacy and reduced side effects.

Silencing circATXN1 in Aging Nucleus Pulposus Cell Alleviates Intervertebral Disc Degeneration via Correcting Progerin Mislocalization.

Circular RNAs (circRNAs) play a critical regulatory role in degenerative diseases; however, their functions and therapeutic applications in intervertebral disc degeneration (IVDD) have not been explored. Here, we identified that a novel circATXN1 highly accumulates in aging nucleus pulposus cells (NPCs) accountable for IVDD. CircATXN1 accelerates cellular senescence, disrupts extracellular matrix organization, and inhibits mitochondrial respiration. Mechanistically, circATXN1, regulated by heterogeneous nuclear ribonucleoprotein A2B1-mediated splicing circularization, promotes progerin translocation from the cell nucleus to the cytoplasm and inhibits the expression of insulin-like growth factor 1 receptor (IGF-1R). To demonstrate the therapeutic potential of circATXN1, siRNA targeting the backsplice junction of circATNX1 was screened and delivered by tetrahedral framework nucleic acids (tFNAs) due to their unique compositional and tetrahedral structural features. Our siRNA delivery system demonstrates superior abilities to transfect aging cells, clear intracellular ROS, and enhanced biological safety. Using siRNA-tFNAs to silence circATXN1, aging NPCs exhibit reduced mislocalization of progerin in the cytoplasm and up-regulation of IGF-1R, thereby demonstrating a rejuvenated cellular phenotype and improved mitochondrial function. In vivo, administering an aging cell-adapted siRNA nucleic acid framework delivery system to progerin pathologically expressed premature aging mice (zmpste24-/-) can ameliorate the cellular matrix in the nucleus pulposus tissue, effectively delaying IVDD. This study not only identified circATXN1 functioning as a cell senescence promoter in IVDD for the first time, but also successfully demonstrated its therapeutic potential via a tFNA-based siRNA delivery strategy.

Gold nanotip array for ultrasensitive electrochemical sensing and spectroscopic monitoring.

A gold nanotip array platform with a combination of ultrasensitive electrochemical sensing and spectroscopic monitoring capability is reported. Adenosine triphosphate is detected down to 1 pM according to the impedance changes in response to aptamer-specific binding. Furthermore, the local molecular information can be monitored at the individual plasmonic nanotips, and hence provide the capability for a better understanding of complex biological processes.

Confirmation of quinolone-induced formation of gyrase-DNA conjugates using AFM.

It was demonstrated in our studies that norfloxacin, a representative member of quinolone antibiotics, can indeed stabilize the gyrase-DNA complex formed during enzymatic cycle. In addition, the formation of the drug-induced complex has been firstly visualized through our atomic force microscopic examination.

Intrinsic curvature in duplex DNA inhibits Human Topoisomerase I.

Human Topoisomerase I (hTopo I) have been known as a potential target for cancer therapy. A series of duplex DNA with different intrinsic curvatures have been designed as inhibitors to hTopo I. The activities of hTopo I on relaxing supercoiled plasmid pUC 19 are apparently diminished in the presence of the curved DNA. More potent inhibitions and smaller IC(50) are achieved by duplex DNA with higher curvatures. EMSA indicates that hTopo I can recognize the curved DNA through binding interactions. Our studies demonstrate that the activity of hTopo I can be modulated by the intrinsic curvature of linear DNA and provide a new avenue to design curved DNA as hTopo I inhibitors with high therapeutic efficiency and low toxicity.

A redox-activated Pt(IV) pro-probe: From G-quadruplex imaging to cancer therapy.

Efficient uptake to both cytoplasm and nucleus in live cells remains a key obstacle for G-quadruplex targeting fluorophores. We developed a Pt(IV) complex by oxidizing a bisphenanthrolinyl Pt(II) complex, which is our first generation G-quadruplex specific fluorogenic probe.15 The axial lipophilic ligand assists Pt(IV) pro-probe to enter live cells and reach the nucleus rapidly. In situ reduction of Pt(IV) pro-probe restores parental Pt(II) complex, and sequentially lights up both RNA and DNA G-quadruplexes in live cancerous cells simultaneously. Pt(IV) pro-probe shows potent cytotoxicity after long time incubation as a dual-functional theranostic agent.

Rapid enrichment and sensitive detection of extracellular vesicles through measuring the phospholipids and transmembrane protein in a microfluidic chip.

Extracellular vesicles (EVs) have attracted tremendous attention in recent years and quantification of EVs is a key issue in the evaluation of vesicle-based diagnostics and therapeutic development, but it's quite challenging to determine whether higher protein expression signals are due to larger vesicle amount or higher protein content within each vesicle. To solve this problem, herein, we proposed a strategy based on staining phospholipid bilayers of EVs with lipophilic dyes to evaluate their lipid amount, which was subsequently normalized as an internal standard for studying the expression of transmembrane protein (i.e., CD63) on EVs in different samples. In addition, a microfluidic platform based on electrophoresis technology was invented to effectively enrich and detect EVs. Small fluorescent labeling molecules (i.e., uncombined aptamers) were on-chip removed from EVs without pre-separation via ultracentrifugation or ultrafiltration which were indispensable in nanoparticle tracking analysis (NTA) and flow cytometry techniques and the performance of this assay is comparable to NTA. Finally, it was found obvious difference in the expression of CD63 on EVs before and after normalization based on lipid amount in plasma samples. This method is expected to provide more accurate information when comparing the expression levels of EVs biomarkers in different samples.