Small Molecule-Based Highly Active and Selective K+ Transporters with Potent Anticancer Activities.

We report here a novel class of cation transporters with extreme simplicity, opening a whole new dimension of scientific research for finding small molecule-based cation transporters for therapeutic applications. Comprising three modular components (a headgroup, a flexible alkyl chain-derived body, and a crown ether-derived foot for ion binding), these transporters efficiently (EC50 = 0.18-0.41 mol % relative to lipid) and selectively (K+/Na+ selectivity = 7.0-9.5) move K+ ions across the membrane. Importantly, the most active (EC50 = 0.18-0.22 mol %) and highly selective series of transporters A12, B12, and C12 concurrently possess potent anticancer activities with IC50 values as low as 4.35 ± 0.91 and 6.00 ± 0.13 µM toward HeLa and PC3 cells, respectively. Notably, a mere replacement of the 18-crown-6 unit in the structure with 12-crown-4 or 15-crown-5 units completely annihilates the cation-transporting ability.

Tricking enzymes in living cells: a mechanism-based strategy for design of DNA topoisomerase biosensors.

Most activity-based molecular probes are designed to target enzymes that catalyze the breaking of chemical bonds and the conversion of a unimolecular substrate into bimolecular products. However, DNA topoisomerases are a class of enzymes that alter DNA topology without producing any molecular segments during catalysis, which hinders the development of practical methods for diagnosing these key biomarkers in living cells. Here, we established a new strategy for the effective sensing of the expression levels and catalytic activities of topoisomerases in cell-free systems and human cells. Using our newly designed biosensors, we tricked DNA topoisomerases within their catalytic cycles to switch on fluorescence and resume new rounds of catalysis. Considering that human topoisomerases have been widely recognized as biomarkers for multiple cancers and identified as promising targets for several anticancer drugs, we believe that these DNA-based biosensors and our design strategy would greatly benefit the future development of clinical tools for cancer diagnosis and treatment.

Cancer Biomarker-Triggered Disintegrable DNA Nanogels for Intelligent Drug Delivery.

Even though various techniques have been developed thus far for targeted delivery of therapeutics, design and fabrication of cancer biomarker-triggered disintegrable nanogels, which are exclusively composed of nucleic acid macromolecules, are still challenging nowadays. Here, we describe for the first time our creation of intelligent DNA nanogels whose backbones are sorely disintegrable by flap endonuclease 1 (FEN1), an enzymatic biomarker that is highly overexpressed in most cancer cells but not in their normal counterparts. It is the catalytic actions of intracellular FEN1 on bifurcated DNA structures that lead to the cancer-specific disintegration of our DNA nanogels and controlled release of drugs in target cancer cells. Consequently, the brand-new strategies introduced in the current report could break new ground in designing drug carriers for eliminating unwanted side effects of chemotherapeutic agents and live-cell probes for cancer risk assessment, diagnosis, and prognosis.

Buckyball-Based Spherical Display of Crown Ethers for De Novo Custom Design of Ion Transport Selectivity.

Searching for membrane-active synthetic analogues that are structurally simple yet functionally comparable to natural channel proteins has been of central research interest in the past four decades, yet custom design of the ion transport selectivity still remains a grand challenge. Here we report on a suite of buckyball-based molecular balls (MBs), enabling transmembrane ion transport selectivity to be custom designable. The modularly tunable MBm-Cn (m = 4-7; n = 6-12) structures consist of a C60-fullerene core, flexible alkyl linkers Cn (i.e., C6 for n-C6H12 group), and peripherally aligned benzo-3m-crown-m ethers (i.e., m = 4 for benzo-12-crown-4) as ion-transporting units. Screening a matrix of 16 such MBs, combinatorially derived from four different crown units and four different Cn linkers, intriguingly revealed that their transport selectivity well resembles the intrinsic ion binding affinity of the respective benzo-crown units present, making custom design of the transport selectivity possible. Specifically, MB4s, containing benzo-12-crown-4 units, all are Li+-selective in transmembrane ion transport, with the most active MB4-C10 exhibiting an EC50(Li+) value of 0.13 µM (corresponding to 0.13 mol % of the lipid present) while excluding all other monovalent alkali-metal ions. Likewise, the most Na+ selective MB5-C8 and K+ selective MB6-C8 demonstrate high Na+/K+ and K+/Na+ selectivity values of 13.7 and 7.8, respectively. For selectivity to Rb+ and Cs+ ions, the most active MB7-C8 displays exceptionally high transport efficiencies, with an EC50(Rb+) value of 105 nM (0.11 mol %) and an EC50(Cs+) value of 77 nM (0.079 mol %).

DNA Binding and Cleavage Modes of Shishijimicin A.

Although shishijimicin A and its extreme potencies against an array of cancer cell lines have been known for more than a decade, its assumed DNA-cleaving mechanism has not been substantiated as yet. Herein we report studies that reveal binding and scission of double-stranded DNA by shishijimicin A. The results of these studies support the proposed hypothesis that DNA strand scissions are caused by 1,4-benzenoid diradicals formed by Bergman cycloaromatization of the enediyne core of shishijimicin A upon activation by thiols. In addition, double-stranded supercoiled DNA-cleavage experiments with shishijimicin A in competition with known minor groove binders, UV spectroscopic studies, and electrophoretic analysis were utilized to clarify the binding mode of the molecule to DNA. These investigations indicate that shishijimicin A binds to the minor groove of double-stranded DNA and that its ß-carboline moiety plays a role in the binding through intercalation. In addition, due to the fact that naked linker regions of DNA in the interphase and metaphase of eukaryotic cells are unprotected by histone proteins during entire cell cycles and because these unprotected regions of DNA are vulnerable to attack by DNA binders, it was concluded that the observed double-strand DNA cleavage and very low sequence selectivity by shishijimicin A may account for its extraordinary cytotoxicity.

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.

Quantitative determination of linking number differences between circular polynucleosomes and histone H1-bound circular polynucleosomes.

With the aim of discovering contribution of histone H1 to linking number changes of DNA, determination of linking number differences between histone H1-free circular polynucleosomes and histone H1-bound circular polynucleosomes was carried out during our investigations. Our results showed that on average, binding of ∼11.5 histone H1 molecules causes one linking number change in circular polynucleosomes in the presence of 1.5 mM spermidine. When concentrations of spermidine decreases or increases, these linking number differences decrease significantly. It is therefore evident that linking number changes caused by histone H1 are spermidine concentration-dependent.

Effects of spermidine and ATP on stabilities of chromatosomes and histone H1-depleted chromatosomes.

It is shown in our FRET studies that both chromatosomes and histone H1-depleted chromatosomes exist in their arm-closed forms in the absence of spermidine. In the presence of spermidine, however, these two types of structural assemblies are converted into their arm-open forms. In addition, ATP as polyanion is capable of suppressing the polycationic effect of spermidine, thus facilitating re-formation of arm-closed forms of these two types of structural assemblies. Our studies therefore illustrate that conversion between arm-closed and arm-open forms of chromatosomes and histone H1-depleted chromatosomes can be manipulated by varying concentrations of polycationic spermidine and polyanionic ATP.

Presence of negative supercoiling in aggregates of histone H1-plasmidic polynucleosome complexes.

It was shown in the past that in the presence of histone H1, plasmidic polynucleosomes formed densely packed aggregates. Our current studies demonstrate that these aggregates are susceptible to the actions of E. coli topoisomerase I, human topoisomerase I and DNA nicking enzyme, which is the indication that negative supercoiling is present in the condensed DNA-protein complexes. Since negative supercoiling leads to formation of highly curved and compact plectonemic and toroidal DNA structures, it would be reasonable to assume that DNA negative supercoils are responsible for aggregation of histone H1-plasmidic polynucleosome complexes.

Computation-guided improved one-pot synthesis of macrocyclic cation-binding aromatic pyridone pentamers.

By using polar DMF to relax the H-bonded rigid backbone and to lower the energetic penalty associated with the sterically-crowded environment, the yields for BOP-mediated one-pot synthesis of pentameric macrocycles can be improved from 10-25% as obtained in CH2Cl2 to 13-47% when 15% DMF in CH2Cl2 was used as the reaction medium.

Investigation of human flap structure-specific endonuclease 1 (FEN1) activity on primer-template models and exploration of a substrate-based FEN1 inhibitor.

Flap structure-specific endonuclease 1 (FEN1) is one of the enzymes that involve in Eukaryotic DNA replication and repair. Recent studies have proved that FEN1 is highly over-expressed in various types of cancer cells and is a drug target. However, a limited number of FEN1 inhibitors has been identified and approved. Herein, we investigate the catalytic activity of FEN1, and propose a substrate-based inhibitor. As a consequence, one of the phosphorothioate-modified substrates is proved to exhibit the most efficient inhibitory effect in our in vitro examinations. A novelly-designed substrate-based FEN1 inhibitor was accordingly constructed and determined a remarkable IC50 value.

Disintegration of cruciform and G-quadruplex structures during the course of helicase-dependent amplification (HDA).

Unlike chemical damages on DNA, physical alterations of B-form of DNA occur commonly in organisms that serve as signals for specified cellular events. Although the modes of action for repairing of chemically damaged DNA have been well studied nowadays, the repairing mechanisms for physically altered DNA structures have not yet been understood. Our current in vitro studies show that both breakdown of stable non-B DNA structures and resumption of canonical B-conformation of DNA can take place during the courses of isothermal helicase-dependent amplification (HDA). The pathway that makes the non-B DNA structures repairable is presumably the relieving of the accumulated torsional stress that was caused by the positive supercoiling. Our new findings suggest that living organisms might have evolved this distinct and economical pathway for repairing their physically altered DNA structures.

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.

Extraordinarily Stable Hairpin-Based Biosensors for Rapid Detection of DNA Ligases.

DNA ligases are essential enzymes involved in DNA replication and repair processes in all organisms. These enzymes seal DNA breaks by catalyzing the formation of phosphodiester bonds between juxtaposed 5' phosphate and 3' hydroxyl termini in double-stranded DNA. In addition to their critical roles in maintaining genomic integrity, DNA ligases have been recently identified as diagnostic biomarkers for several types of cancers and recognized as potential drug targets for the treatment of various diseases. Although DNA ligases are significant in basic research and medical applications, developing strategies for efficiently detecting and precisely quantifying these crucial enzymes is still challenging. Here, we report our design and fabrication of a highly sensitive and specific biosensor in which a stable DNA hairpin is utilized to stimulate the generation of fluorescence signals. This probe is verified to be stable under a wide range of experimental conditions and exhibits promising performance in detecting DNA ligases. We anticipate that this hairpin-based biosensor will significantly benefit the development of new targeting strategies and diagnostic tools for certain diseases.

Ultrasensitive colorimetric DNA detection using a combination of rolling circle amplification and nicking endonuclease-assisted nanoparticle amplification (NEANA).

A combination of rolling circle amplification and nicking endonuclease-assisted nanoparticle amplification (NEANA) is used for the rapid, colorimetric detection of DNA. The integration of rolling circle amplification into the NEANA approach allows for detection of oligonucleotides with arbitrary sequences at ultralow concentrations.

Fluorescence and visual detection of single nucleotide polymorphism using cationic conjugated polyelectrolyte.

We report a simple assay for visual detection of single nucleotide polymorphisms (SNPs) with good sensitivity and selectivity. The selectivity is determined by Escherichia coli (E. coli) DNA ligase mediated circular formation upon recognition of the point mutation on DNA targets. Rolling cycle amplification (RCA) of the perfect-matched DNA target is then initiated using the in situ formed circular template in the presence of Phi29 enzyme. Due to amplification of the DNA target, the RCA product has a tandem-repeated sequence, which is significantly longer than that for the SNP strand. Direct addition of a cationic conjugated polymer of poly[9,9'-bis(6'-(N,N,N-trimethylammonium)hexyl)fluorene-co-9,9'-bis(2-(2-(2-(N,N,N-trimethylammonium)ethoxyl)-ethoxy)-ethyl)fluorene tetrabromide] containing 20 mol% 2,1,3-benzothiadiazole (PFBT(20)) into the RCA solution leads to blue-whitish fluorescent color for SNP strand and yellowish fluorescent color for amplified DNA, due to PFBT(20)/DNA complexation induced intrachain/interchain energy transfer. To further improve the contrast for visual detection, FAM-labeled peptide nucleic acid (PNA) was hybridized to each amplified sequence, which is followed by the addition of poly{2,7-[9,9-bis(6'-N,N,N-trimethylammoniumhexyl)]fluorene-co-2,5-difluoro-1,4-phenylene dibromide} (PFP). The PNA/DNA hybridization brings PFP and FAM-PNA into close proximity for energy transfer, and the solution fluorescent color appears green in the presence of target DNA with a detection limit of 1 nM, which is significantly improved as compared to that for most reported visual SNP assay.

Observation of backbone self-crossings of organismal DNAs through atomic force microscopy.

It is demonstrated in our studies that intrinsic curvatures of organismal DNAs could lead to the generation of duplex backbone self-crossings in their relaxed forms. In addition, the backbone self-crossings of the organismal DNAs could be readily confirmed through 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.

Interference of intrinsic curvature of DNA by DNA-intercalating agents.

It has been demonstrated in our studies that the intrinsic curvature of DNA can be easily interrupted by low concentrations of chloroquine and ethidium bromide. In addition, the changes of DNA curvature caused by varying the concentration of these two DNA intercalators can be readily verified through using an atomic force microscope.

Conformational organizations of G-quadruplexes composed of d(G(4)T(n))(3)G(4).

Structural polymorphism is one of the important issues with regard to G-quadruplexes because the structural diversity may significantly affect their biological functions in vivo and their physical property in nano-material. A series of oligonucleotides with four repeat guanines sequence [d(G(4)T(n))(3)G(4) (n=1-6)] were designed. In this study, the effects of loop length on the formation of structures of G-quadruplex were investigated through the result of CD (circular dichroism) and 20% non-denatured polyacrylamide gel electrophoresis. Our studies demonstrate that the length of loop in 100mM KCl solution could predict the conformation of G-quadruplex.