Recovery of the Formation and Function of Oxidized G-Quadruplexes by a Pyrene-Modified Guanine Tract.

Oxidation is one of the frequent causes of DNA damage, especially to guanine bases. Guanine bases in the G-quadruplex (G4) are sensitive to damage by oxidation, resulting in transformation to 8-oxo-7,8-dihydroguanine (8-oxoG). Because the formation of G4 represses the expression of some cancer-related genes, the presence of 8-oxoG in a G4 sequence might affect G4 formation and induce cancer progression. Thus, oxidized-G4 formation must be controlled using a chemical approach. In the present study, we investigated the effect of introduction of 8-oxoG into a G4 sequence on the formation and function of the G4 structure. The 8-oxoG-containing G4 derived from the promoter region of the human vascular endothelial growth factor ( VEGF) gene differed topologically from unoxidized G4. The oxidized VEGF G4 did not act as a replication block and was not stabilized by the G4-binding protein nucleolin. To recover G4 function, we developed an oligonucleotide consisting of a pyrene-modified guanine tract that replaces the oxidized guanine tract and forms stable intermolecular G4s with the other intact guanine tracts. When this oligonucleotide was used, the oxidized G4 stalled replication and was stabilized by nucleolin as with the unmodified G4. This strategy generally enables recovery of the function of any oxidized G4s and therefore has potential for cancer therapy.

Facile conversion of ATP-binding RNA aptamer to quencher-free molecular aptamer beacon.

We have developed RNA-based quencher-free molecular aptamer beacons (RNA-based QF-MABs) for the detection of ATP, taking advantage of the conformational changes associated with ATP binding to the ATP-binding RNA aptamer. The RNA aptamer, with its well-defined structure, was readily converted to the fluorescence sensors by incorporating a fluorophore into the loop region of the hairpin structure. These RNA-based QF-MABs exhibited fluorescence signals in the presence of ATP relative to their low background signals in the absence of ATP. The fluorescence emission intensity increased upon formation of a RNA-based QF-MAB·ATP complex.

Design and Properties of Ligand-Conjugated Guanine Oligonucleotides for Recovery of Mutated G-Quadruplexes.

The formation of a guanine quadruplex DNA structure (G4) is known to repress the expression of certain cancer-related genes. Consequently, a mutated G4 sequence can affect quadruplex formation and induce cancer progression. In this study, we developed an oligonucleotide derivative consisting of a ligand-containing guanine tract that replaces the mutated G4 guanine tract at the promoter of the vascular endothelial growth factor (VEGF) gene. A ligand moiety consisting of three types of polyaromatic hydrocarbons, pyrene, anthracene, and perylene, was attached to either the 3' or 5' end of the guanine tract. Each of the ligand-conjugated guanine tracts, with the exception of anthracene derivatives, combined with other intact guanine tracts to form an intermolecular G4 on the mutated VEGF promoter. This intermolecular G4, exhibiting parallel topology and high thermal stability, enabled VEGF G4 formation to be recovered from the mutated sequence. Stability of the intramolecular G4 increased with the size of the conjugated ligand. However, suppression of intermolecular G4 replication was uniquely dependent on whether the ligand was attached to the 3' or 5' end of the guanine tract. These results indicate that binding to either the top or bottom guanine quartet affects unfolding kinetics due to polarization in DNA polymerase processivity. Our findings provide a novel strategy for recovering G4 formation in case of damage, and fine-tuning processes such as replication and transcription.

Quencher-free molecular aptamer beacons (QF-MABs) for detection of ATP.

We have constructed a simple and efficient system-based on quencher-free molecular aptamer beacons (QF-MABs)-for probing ATP. In the absence of ATP, the fluorescence of a pyrene fluorophore on the loop position (15 nucleotides from the 5' end) of the optimal QF-MAB was quenched by the neighboring nucleobases; in its presence, fluorescence was recovered, due to a conformational change in the secondary structure of the QF-MAB.

Photophysical and structural investigation of a (Py)A-modified adenine cluster: its potential use for fluorescent DNA probes exhibiting distinct emission color changes.

In this study, we found a (Py)A-modified adenine cluster (A-cluster), a minimum fluorescent unit for significant emission wavelength changes, and investigated its photophysical and structural properties. The basic A-cluster unit was an adenine-pentad duplex containing stacked (Py)A pairs in the center aligned in an antiparallel manner. Spectral analysis of the A-cluster revealed remarkable reddish fluorescence with a large Stokes shift (∼195 nm) and a long life-time constant (31 ns), originated from exciton states formed by (Py)A pairs and neighboring adenines. Structurally, the exciton state of the A-cluster exhibited unusually high stability, relative to that of other five-mismatched duplexes, as a result of stabilization through strong stacking interactions (zipper-like structure) of the mismatched A-A and (Py)A pairs, rather than through traditional Watson-Crick base pairing. These spectral and structural properties of the A-clusters were specific to the adenine bases and highly disturbed by introducing other bases (T, G, and especially C) or an abasic site into the A-cluster, whereas they were enhanced through synergistic effects in systems containing multiple A-clusters. As a minimum unit for these unique properties, finally, the A-cluster was exploited as a fluorescent probing system for specific nucleic acid sequences, such as miR-21, accompanying distinct fluorescence color changes from blue to red. These findings indicated the potential utility of the A-cluster as a part of fluorescent probes exhibiting clear signaling upon micro-environmental changes.

Cancer-specific gene silencing through therapeutic siRNA delivery with B vitamin-based nanoassembled low-molecular-weight hydrogelators.

This paper describes the synthesis, characterization, and in vitro and in vivo siRNA transfection ability of B vitamin-based cationic clickable bolaamphiphiles (VBs). Our VBs derived from vitamins B2, B3, B5, B6, and B7 formed nanoassembled low-molecular-weight hydrogelators (LMWGs, vitagels). The vitagels VB2, VB6, and VB7 (derived from vitamins B2, B6, and B7, respectively) facilitated delivery of small interfering RNAs (siRNA), efficiently silencing gene expression specifically into cancer cell lines; in addition, the LMWGs derived from vitamins B3, B5, and B6 were biocompatible. An ex vivo study in a mouse model revealed that the siRNA delivered by the vitagel VB7 was located primarily at the site of the tumor. The gene silencing efficiency of vascular endothelial growth factor siRNA delivered by vitagels was dependent on the nature of the vitamin headgroup, the N/P ratio, and, interestingly, the hydrogelation properties of the VBs.

5'-Triphosphate-RNA-independent activation of RIG-I via RNA aptamer with enhanced antiviral activity.

RIG-I is a cytosolic receptor for non-self RNA that mediates immune responses against viral infections through IFNα/ß production. In an attempt to identify novel tools that modulate IFNα/ß production, we used SELEX technology to screen RNA aptamers that specifically target RIG-I protein. Most of the selected RIG-I aptamers contained polyU motifs in the second half regions that played critical roles in the activation of RIG-I-mediated IFNß production. Unlike other known ligands, RIG-I aptamer bound and activated RIG-I in a 5'-triphosphate-independent manner. The helicase and RD domain of RIG-I were used for aptamer binding, but intact RIG-I protein was required to exert aptamer-mediated signaling activation. Furthermore, replication of NDV, VSV and influenza virus in infected host cells was efficiently blocked by pre- or post-treatment with RIG-I aptamer. Based on these data, we propose that RIG-I aptamer has strong potential to be an antiviral agent that specifically boosts the RIG-I-dependent signaling cascade.

(DNS)C: a fluorescent, environmentally sensitive cytidine derivative for the direct detection of GGG triad sequences.

With the goal of developing a fluorescent nucleoside sensitive to its environment, in this study we synthesized (DNS)C, a novel modified 2'-deoxycytidine bearing a 5-(dimethylamino)naphthalene-1-sulfonyl (dansyl) moiety at the N4 position, and tested its properties in monomeric and oligomeric states. (DNS)C undergoes intramolecular photoinduced electron transfer between its dansyl and cytosine units, resulting in remarkable changes in fluorescence that depend on the choice of solvent. In addition, the fluorescence behavior and thermal stability of oligonucleotides containing (DNS)C are dependent on the nature of the flanking and neighboring bases. Notably, (DNS)C exhibits fluorescence enhancement only in fully matched duplex DNA containing a GGG triad sequence. The environmental sensitivity of (DNS)C can be exploited as a fluorescence tool for monitoring the interactions of DNA with other biomolecules, including DNA, RNA, and proteins.

Delivery of floxuridine derivatives to cancer cells by water-soluble organometallic cages.

The self-assembly of 2,4,6-tris(pyridin-4-yl)-1,3,5-triazine (tpt) triangular panels with p-cymene (pPr(i)C(6)H(4)Me) ruthenium building blocks and 2,5-dioxydo-1,4-benzoquinonato (dobq) or 5,8-dioxydo-1,4-naphthoquinonato (donq) bridges, in the presence of a pyrenyl-nucleoside derivatives (pyreneR), affords the triangular prismatic host-guest compounds [(pyrene-R)⊂Ru(6)(pPr(i)C(6)H(4)Me)(6)(tpt)(2)(dobq)(3)](6+) ([(pyrene-R)⊂1](6+)) and [(pyrene-R)⊂Ru(6)(pPr(i)C(6)H(4)Me)(6)(tpt)(2)(donq)(3)](6+) ([(pyrene-R)⊂2](6+)), respectively. The inclusion of six monosubstitutedpyrenyl-nucleosides (pyrene-R1 = 5'-(1-pyrenyl butanoate)-2'-deoxyuridine, pyrene-R2 = 5-fluoro-5'-(1-pyrenyl butanoate)-2'-deoxyuridine, pyrene-R3 = 5'-{N-[1-oxo-4-(1-pyrenyl)butyl]-glycyl}-2'-deoxyuridine, pyrene-R4 = 5-fluoro-5'-{N-[1-oxo-4-(1-pyrenyl)butyl]-glycyl}-2'-deoxyuridine, pyrene-R5 = 5-fluoro-5'-{N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanyl}-2'-deoxyvuridine, pyrene-R6 = 5-fluoro-5'-{N-[1-oxo-4-(1-pyrenyl)butyl]-phenylalanyl}-2'-deoxyuridine) has been accomplished. The carceplex nature of [(pyrene-R)⊂1](6+) with the pyrenyl moiety firmly encapsulated in the hydrophobic cavity of the cage with the nucleoside groups pointing outward was confirmed by NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS), while the host-guest nature of [(pyrene-R)⊂2](6+) was studied in solution by NMR techniques. In contrast to the floxuridine compounds used in the clinic, the host-guest complexes are highly water-soluble. Consequently, the cytotoxicities of these water-soluble compounds have been established using human ovarian A2780 and A2780cisR cancer cells. All the host-guest systems are more cytotoxic than the empty cages alone [1][CF(3)SO(3)](6) (IC(50) = 23 µM) and [2][CF(3)SO(3)](6) (IC(50) = 10 µM), the most active compound [pyrene-R4⊂1][CF(3)SO(3)](6)being 2 orders of magnitude more cytotoxic (IC(50) = 0.3 µM) on these human ovarian cancer cell lines (A2780 and A2780cisR).

Complexation and conjugation approaches to evaluate siRNA delivery using cationic, hydrophobic and amphiphilic peptides.

In this study, we used solid phase synthesis to prepare three kinds of peptides and then formulated their peptide-siRNA complexes and peptide-siRNA conjugates. Both the complexation and conjugation systems were nontoxic and allowed the delivery of siRNA into the cytoplasm without the need for any transfection agents and with subsequent inhibition of gene expression.

Enhancing Peptide Nucleic Acid-Nanomaterial Interaction and Performance Improvement of Peptide Nucleic Acid-Based Nucleic Acid Detection by Using Electrostatic Effects.

Single-stranded peptide nucleic acid (PNA) probes interact strongly with several nanomaterials, and the interaction was diminished in the presence of complementary nucleic acid targets which forms the basis of many nucleic acid sensing platforms. As opposed to the negatively charged DNA probes, the charges on the PNA probes may be fine-tuned by incorporating amino acids with charged side chains. The contribution of electrostatic effects to the interaction between PNA probes and nanomaterials has been largely overlooked. This work reveals that electrostatic effects substantially enhanced the quenching of dye-labeled conformationally constrained pyrrolidinyl PNA probes by several nanomaterials including graphene oxide (GO), reduced graphene oxide, gold nanoparticles (AuNPs), and silver nanoparticles. The fluorescence quenching and the color change from red to purple in the case of AuNPs because of aggregation were inhibited in the presence of complementary nucleic acid targets. Thus, fluorescence and colorimetric assays for DNA and RNA that can distinguish even single-base-mismatched nucleic acids with improved sensitivity over conventional DNA probes were established. Both the GO- and AuNP-based sensing platforms have been successfully applied for the detection of real DNA and RNA samples in vitro and in living cells. This study emphasizes the active roles of electrostatic effects in the PNA-nanomaterial interactions, which paves the way toward improving the performance of PNA-nanomaterial based assays of nucleic acids.

Quencher-free molecular beacon: Enhancement of the signal-to-background ratio with graphene oxide.

We report the highly improved version of quencher-free molecular beacon (QF-MB) system by using graphene oxide (GO) as an external quencher. This QF-MB/GO system provided a higher S/B ratio (31.0) relative to that (2.2) of the same system in the absence of GO, while retaining a high selectivity for fully matched over single-base-mismatched targets.

pH-responsive self-duplex of (Py)A-substituted oligodeoxyadenylate in graphene oxide solution as a molecular switch.

In this paper, we demonstrated a highly discriminated and reliable molecular switch based on the interaction between the self-duplex of (Py)A-substituted oligodeoxyadenylate and graphene oxide in aqueous solution. This system showed a clear on/off state through the association and dissociation of (Py)A-modified oligodeoxynucleotide with graphene oxide in manipulated pH conditions, high amplitude efficiency for at least 50 cycles, and rapid response within seconds. Our molecular switch system has high reproducibility and simple operation by using pH stimulus.

Cationic nucleolipids as efficient siRNA carriers.

We synthesized five novel uridine-based cationic nucleolipids, introducing basic amino acid residues at the 5' position of uridine, through 1,3-dipolar cycloaddition, and hydrophobic alkyl moieties at the 2' and 3' positions, through carbamate linkages. Their lipoplexes delivered siRNAs efficiently to cells, in vitro, without any severe toxicity.

Pyrene-Modified Guanine Cluster Probes Forming DNA/RNA Hybrid Three-Way Junctions for Imaging of Intracellular MicroRNAs.

MicroRNAs (miRNAs) regulate gene expression in cells; high levels of expression are associated with various cancers. In this paper, we describe PyA-modified nucleic acid probes that can detect intracellular miRNAs by forming DNA/RNA hybrid three-way junction structures containing a fluorescent scaffold-a so-called G-cluster. This G-cluster featured two mismatched strands, four guanine residues, and one fluorescent adenine residue having a pyrene moiety covalently connected at the 8-position through an acetylene linker. The scaffold underwent a dramatic shift in its emission wavelength when two mismatched strands formed a duplex, similar to the behavior of an adenine pentad system (A-cluster). We applied the G-cluster scaffold in a three-way junction system to probe for miRNAs; its red-shifted fluorescence intensity and stability were greater than those reported previously for A-cluster three-way junction probes. Furthermore, confocal microscopy of cancer cell lines revealed bright fluorescence emissions in response to the miRNAs in the cells. Thus, this system can be applied intracellularly as a potential fluorescent probe for the detection of various biologically important nucleic acids.

Label-free impedimetric sensor for a ribonucleic acid oligomer specific to hepatitis C virus at a self-assembled monolayer-covered electrode.

A ribonucleic acid (RNA) sensor based on hybridization of its peptide nucleic acid (PNA) molecule with a target RNA oligomer of the internal ribosome entry site sequence specific to the hepatitis C virus (HCV) and the electrochemical impedance detection is described. This RNA is one of the most conservative molecules of the whole HCV RNA genome. The ammonium ion terminated PNA molecule was immobilized via its host-guest interactions with the diaza crown ring of 3-thiophene-acetamide-diaza-18-crown-6 synthesized by a simple two-step method, which forms a well-defined self-assembled monolayer (SAM) on gold. Hybridization events of the probe PNA with the target RNA were monitored by measuring charge-transfer resistances for the Fe(CN)(6)(3-/4-) redox probe using Fourier transform electrochemical impedance spectroscopy. The ratio of the resistances of the SAM-covered electrode measured before and after hybridization increased linearly with log[RNA] in the rat liver lysate with a detection limit of about 23 pM.

Design, synthesis, and anticancer activities of novel perfluoroalkyltriazole-appended 2'-deoxyuridines.

We have focused on the C5-modification of 2'-deoxyuridine with substituted heterocycles for bioactivity, such as antiviral or anticancer activity. Herein, we report a novel class of nucleoside analogues with perfluoroalkyltriazole moiety as an anticancer drug candidate.

Label-free electrochemical detection of the p53 core domain protein on its antibody immobilized electrode.

We report quantitative results on interactions between a tumor suppressor protein, p53, also known as a prognostic cancer marker, and its antibody. The p53 antibody molecules immobilized on an (R)-lipo-diaza-18-crown-6 self-assembled monolayer (SAM)-modified gold disk electrode were shown to effectively capture the p53 protein by Western blot, quartz crystal microbalance, and electrochemical impedance experiments. The p53 protein thus captured modulated the ability of the electrode for charge transfer to and from a redox probe in the solution in a p53 concentration range of approximately 0.1-30 microg/mL. The same interaction was also observed in the human embryonic kidney cell lysate, demonstrating that the SAM-modified electrode can serve as a selective platform for electrochemically monitoring the cellular p53 concentration.

(R)-lipo-diaza-18-crown-6 self-assembled monolayer as a selective serotonin receptor.

A highly selective receptor for serotonin was designed using cages formed by the (R)-lipo-diaza-18-crown-6 self-assembled monolayer (SAM) on gold and experimentally verified by a variety of electrochemical experiments in solutions containing large amounts of dopamine and ascorbic acid, as well as other interferents. The molecular modeling study showed that parameters such as the H-pi interaction provided important driving forces for the cage to form a strong inclusion complex with serotonin. The charge-transfer resistance (R(CT)'s) to/from redox probe ions, Fe(CN)(6)(3-/4-), was greatly enhanced because of their electrostatic attractions to ammonium ions of serotonin molecules captured by cages. The changes in R(CT)-values were shown to be remarkably selective for serotonin in the presence of many interferents.

Synthesis of 5-isoxazol-5-yl-2'-deoxyuridines exhibiting antiviral activity against HSV and several RNA viruses.

This paper describes a simple method for synthesizing a small library of 5-isoxazol-5-yl-2'-deoxyuridines from 5-iodo-2'-deoxyuridine. Nitrile oxides were generated in situ from oximes using a commercial bleaching agent; their cycloaddition with 5-ethynyl-2'-deoxyuridine yielded isoxazoles possessing activity against herpes simplex viruses 1 and 2, Encephalomyocarditis virus, Coxsackie B3, and vesicular stomatitis virus; these isoxazoles were, however, inactive against corona virus, influenza virus, and HIV.