Progress and Prospects of Pyrrole‐Imidazole Polyamide–Fluorophore Conjugates as Sequence‐Selective DNA Probes

Recently, the versatility of N‐methylpyrrole (Py)‐N‐methylimidazole (Im) polyamide conjugates, which have been developed from the DNA‐binding antibiotics distamycin A and netropsin, has been shown. These synthetic small molecules can permeate cells to bind with duplex DNA in a sequence‐specific manner, and hence can influence gene expression in vivo. Accordingly, several reports demonstrating the sequence specificity and biological activity of Py‐Im polyamides have accumulated. However, the benefits of Py‐Im polyamides, in particular those conjugated with fluorophores, has been overlooked. Moreover, clear directions for the employment of these attractive artificial small molecules have not yet been shown. Here, we present a detailed overview of the current and prospective applications of Py‐Im polyamide–fluorophore conjugates, including sequence‐specific recognition with fluorescence emission properties, and their potential roles in biological imaging.     

Label-Free in Situ Monitoring of the DNA Hybridization Chain Reaction by Using Sequence-Selective Minor-Groove-Binding Fluorophores

A new label-free in situ monitoring system for the hybridization chain reaction (HCR) based on DNA minor-groove-binding fluorophores [Hoechst 33258 (Hoe) or quinone cyanine-dithiazole (QCy-DT)] has been developed. Use of two unmodified hairpin oligodeoxyribonucleotides containing incomplete double-stranded AATT sequences enabled target-dependent formation of probe binding sites—that is, AATT double strand—in the HCR product, together with fluorescence enhancement of minor-groove-binding fluorophores in situ. This system allows target DNA to be detected through the fluorescence enhancement of Hoe and QCy-DT in real time and in situ. Further development of a label-free, isothermal detection system might provide a cost-effective and user-friendly method for nucleic acid detection.     

Detection of HER2+ Breast Cancer Cells using Bioinspired DNA-Based Signal Amplification

Circulating tumor cells (CTC) are promising biomarkers for metastatic cancer detection and monitoring progression. However, detection of CTCs remains challenging due to their low frequency and heterogeneity. Herein, we report a bioinspired approach to detect individual cancer cells, based on a signal amplification cascade using a programmable DNA hybridization chain reaction (HCR) circuit. We applied this approach to detect HER2⁺ cancer cells using the anti-HER2 antibody (trastuzumab) coupled to initiator DNA eliciting a HCR cascade that leads to a fluorescent signal at the cell surface. At 4 °C, this HCR detection scheme resulted in highly efficient, specific and sensitive signal amplification of the DNA hairpins specifically on the membrane of the HER2⁺ cells in a background of HER2⁻ cells and peripheral blood leukocytes, which remained almost non-fluorescent. The results indicate that this system offers a new strategy that may be further developed toward an in vitro diagnostic platform for the sensitive and efficient detection of CTC.     

Live‐Cell MicroRNA Imaging through MnO2 Nanosheet‐Mediated DD‐A Hybridization Chain Reaction

Innovative techniques to visualize native microRNAs (miRNAs) in live cells can dramatically impact current research on the roles of miRNA in biology and medicine. Here, we report a novel approach for live‐cell miRNA imaging using a biodegradable MnO₂ nanosheet‐mediated DD‐A FRET hybridization chain reaction (HCR). The MnO₂ nanosheets can adsorb DNA hairpin probes and deliver them into live cells. After entering cells, the MnO₂ nanosheets are degraded by cellular GSH. Then, the target miR‐21 triggers cascaded assembly of the liberated hairpin probes into long dsDNA polymers, which brings each two FAMs (d onor) and one TAMRA (a cceptor) into close proximity to generate significantly enhanced DD‐A FRET signals, which was discovered and proven by our previous report. We think the developed approach can serve as an excellent intracellular miRNAs detection tool, which promises the potential for biological and disease studies.     

Synthesis and biological properties of sequence-specific DNA-alkylating pyrrole-imidazole polyamides

In recent years, many diseases including cancer and hereditary and viral diseases have been understood at the DNA sequence level. Direct control of the expression level of a specific gene would provide a promising approach for knowledge-based therapy. N-Methylpyrrole (Py) and N-methylimidazole (Im) polyamides are a new type of small compound that precisely bind to the minor groove of the DNA duplex in a sequence-specific fashion and recruit alkylating agents to the target sequence. We designed and synthesized a series of sequence-specific alkylating Py-Im polyamide conjugates that selectively alkylate predetermined DNA sequences. We have demonstrated that sequence-specific alkylating agents possess gene-silencing activities and a promising potency against human cancer cell lines as well as against xenografts of human cancer cell lines. In this Account, we focus on recent progress in alkylating Py-Im polyamides with regard to sequence specificity and biological activities and the future direction of rational molecular design of genetic switches in the postgenome era is described.     

Tetraphenylethene Derivatives Modulate the RNA Hairpin-G-Quadruplex Conformational Equilibria in Proto-oncogenes**

RNA G-quadruplex (GQs) sequences in 5′-UTRs of certain proto-oncogenes co-localize with hairpin (Hp) forming sequences resulting in intramolecular Hp-GQ conformational equilibria, which is suggested to regulate cancer development and progression. Thus, regulation of Hp-GQ equilibria with small molecules is an attractive but less explored therapeutic approach. Herein, two tetraphenylethene (TPE) derivatives, TPE−Py and TPE-MePy, were synthesized and their effect on Hp-GQ equilibrium was explored. FRET, CD and molecular docking experiments suggest that cationic TPE-MePy shifts the Hp-GQ equilibrium significantly towards the GQ conformer mainly through π-π stacking and van der Waals interactions. In the presence of TPE-MePy, the observed rate constant values for first and second folding steps were increased up to 14.6 and 2.6-fold, respectively. The FRET melting assay showed a strong stabilizing ability of TPE-MePy (ΔTm=4.36 °C). Notably, the unmethylated derivative TPE−Py did not alter the Hp-GQ equilibrium. Subsequently, luciferase assay analysis demonstrated that the TPE-MePy derivatives suppressed the translation efficiency by ∼5.7-fold by shifting the Hp-GQ equilibrium toward GQ conformers in the 5’-UTR of TRF2. Our data suggests that HpGQ equilibria could be selectively targeted with small molecules to modulate translation for therapy.     

A Mutation-Based Method for Pinpointing a DNA N6-Methyladenine Methyltransferase Modification Site at Single Base Resolution

DNA N⁶-methyladenine (6mA) has recently received notable attention due to an increased finding of its functional roles in higher eukaryotes. Here we report an enzyme-assisted chemical labeling method to pinpoint the DNA 6mA methyltransferase (MTase) substrate modification site at single base resolution. A designed allyl-substituted MTase cofactor was applied in the catalytic transfer reaction, and the allyl group was installed to the N⁶-position of adenine within a specific DNA sequence to form N⁶-allyladenine (6aA). The iodination of 6aA allyl group induced the formation of 1, N⁶-cyclized adenine which caused mutations during DNA replication by a polymerase. Thus the modification site could be precisely detected by a mutation signal. We synthesized 6aA deoxynucleoside and deoxynucleotide model compounds and a 6aA-containing DNA probe, and screened nine DNA polymerases to define an optimal system capable of detecting the substrate modification site of a DNA 6mA MTase at single-base resolution.     

N4-Allyldeoxycytidine: A New DNA Tag with Chemical Sequencing Power for Pinpointing Labelling Sites,Mapping Epigenetic Markers,and In Situ Imaging

DNA tagging with base analogues has found numerous applications. To precisely record the DNA labelling information, it would be highly beneficial to develop chemical sequencing tags that can be encoded into DNA as regular bases and decoded as mutant bases following a mild, efficient and bioorthogonal chemical treatment. Here we reported such a DNA tag, N⁴-allyldeoxycytidine (a⁴dC), for labeling and identifying DNA by in vitro assays. The iodination of a⁴dC led to fast and complete formation of 3 , N⁴-cyclized deoxycytidine, which induced base misincorporation during DNA replication and thus could be located at single base resolution. We explored the applications of a⁴dC in pinpointing DNA labelling sites at single base resolution, mapping epigenetic marker N⁴-methyldeoxycytidine, and imaging nucleic acids in situ. In addition, mammalian cellular DNA could be metabolically labelled with a⁴dC. Our study sheds light on the design of next generation DNA tags with chemical sequencing power.     

An Isocytidine Derivative with a 2‐Amino‐6‐methylpyridine Unit for Selective Recognition of the CG Interrupting Site in an Antiparallel Triplex DNA

Sequence‐specific recognition of duplex DNA mediated by triple helix formation offers a potential basis for oligonucleotide therapy and biotechnology. However, triplex formation is limited mostly to homopurine strands, due to poor stabilization at CG or TA base pairs in the target duplex DNA sequences. Several non‐natural nucleosides have been designed for the recognition of CG or TA base pairs within an antiparallel triplex DNA. Nevertheless, problems including low selectivity and high dependence on the neighboring bases remain unsolved. We thus synthesized N²‐arylmethyl isodC derivatives and incorporated them into triplex‐forming oligonucleotides (TFOs) for the selective recognition of the CG base pair within antiparallel triplex DNA. It was shown that an isodC derivative bearing a 2‐amino‐6‐methylpyridine moiety (AP‐isodC) recognizes the CG base pair with high selectivity in antiparallel triplex DNA irrespective of the flanking base pairs.     

Single Labeled DNA FIT Probes for Avoiding False‐Positive Signaling in the Detection of DNA/RNA in qPCR or Cell Media

Oligonucleotide hybridization probes that fluoresce upon binding to complementary nucleic acid targets allow the real‐time detection of DNA or RNA in homogeneous solution. The most commonly used probes rely on the distance‐dependent interaction between a fluorophore and another label. Such duallabeled oligonucleotides signal the change of the global conformation that accompanies duplex formation. However, undesired nonspecific binding events and/or probe degradation also lead to changes in the label–label distance and, thus, to ambiguities in fluorescence signaling. Herein, we introduce singly labeled DNA probes, “DNA FIT probes”, that are designed to avoid false‐positive signals. A thiazole orange (TO) intercalator dye serves as an artificial base in the DNA probe. The probes show little background because the attachment mode hinders 1) interactions of the “TO base” in cis with the disordered nucleobases of the single strand, and 2) intercalation of the “TO nucleotide” with double strands in trans. However, formation of the probe–target duplex enforces stacking and increases the fluorescence of the TO base. We explored open‐chain and carbocyclic nucleotides. We show that the incorporation of the TO nucleotides has no effect on the thermal stability of the probe–target complexes. DNA and RNA targets provided up to 12‐fold enhancements of the TO emission upon hybridization of DNA FIT probes. Experiments in cell media demonstrated that false‐positive signaling was prevented when DNA FIT probes were used. Of note, DNA FIT probes tolerate a wide range of hybridization temperature; this enabled their application in quantitative polymerase chain reactions.     

Carbohydrate–PNA and Aptamer–PNA Conjugates for the Spatial Screening of Lectins and Lectin Assemblies

Nucleic acid architectures offer intriguing opportunities for the interrogation of structural properties of protein receptors. In this study, we performed a DNA‐programmed spatial screening to characterize two functionally distinct receptor systems: 1) structurally well‐defined Ricinus communis agglutinin (RCA₁₂₀), and 2) rather ill‐defined assemblies of L‐selectin on nanoparticles and leukocytes. A robust synthesis route that allowed the attachment both of carbohydrate ligands—such as N‐acetyllactosamine (LacNAc), sialyl‐Lewis‐X (sLe^X), and mannose—and of a DNA aptamer to PNAs was developed. A systematically assembled series of different PNA–DNA complexes served as multivalent scaffolds to control the spatial alignments of appended lectin ligands. The spatial screening of the binding sites of RCA₁₂₀ was in agreement with the crystal structure analysis. The study revealed that two appropriately presented LacNAc ligands suffice to provide unprecedented RCA₁₂₀ affinity (K_D=4 μM ). In addition, a potential secondary binding site was identified. Less dramatic binding enhancements were obtained when the more flexible L‐selectin assemblies were probed. This study involved the bivalent display both of the weak‐affinity sLe^X ligand and of a high‐affinity DNA aptamer. Bivalent presentation led to rather modest (sixfold or less) enhancements of binding when the self‐assemblies were targeted against L‐selectin on gold nanoparticles. Spatial screening of L‐selectin on the surfaces of leukocytes showed higher affinity enhancements (25‐fold). This and the distance–activity relationships indicated that leukocytes permit dense clustering of L‐selectin.     

DNA Interstrand Crosslinks by H‐pin Polyamide (S)‐seco‐CBI Conjugates

Although DNA interstrand crosslinking (ICL) agents are widely used as antitumor drugs, DNA sequence‐specific ICL agents are quite rare. In this study, H‐pin imidazole‐pyrrole polyamide 1‐(chloromethyl)‐2,3‐dihydro‐1H‐benzo[e]indol‐5‐ol (seco‐CBI) conjugates that produce sequence‐specific DNA ICLs were designed and synthesized. Conjugates with H‐pin polyamide and seco‐CBI moieties were constructed to recognize a 7 bp DNA sequence, and their reactivity and selectivity in DNA alkylation were evaluated by using high‐resolution denaturing gel electrophoresis and sequence‐specific plasmid cleavage. One conjugate ( 6 ), which contained a chiral (S)‐seco‐CBI, exhibited greater sequence‐specific ICL activity toward the target DNA sequence and was cytotoxic to a cancer cell line. Molecular modeling studies indicated that the greater activity of 6 resulted from the relative orientation of the cyclopropane group in the (S)‐CBI unit.     

Ferrocene clicked polypyrrole derivatives: effect of spacer group on electrochemical properties and post-polymerization functionalization

In this study, novel ferrocene-functionalized N-alkyl substituted pyrrole derivatives, namely 4-ferrocenyl-1-[3-(pyrrol-1-yl)propyl]-1H-1,2,3-triazole (Py3Fc), 4-ferrocenyl-1-[4-(pyrrol-1-yl)butyl]-1H-1,2,3-triazole (Py4Fc), and 4-ferrocenyl-1-[6-(pyrrol-1-yl)hexyl]-1H-1,2,3-triazole (Py6Fc), were synthesized via click reaction and the monomers were characterized by ¹H NMR, ¹³C NMR, FTIR, and HRMS techniques. Redox properties of the monomers were investigated by cyclic voltammetry (CV) studies. Contrary to general literature, both Py4Fc and Py6Fc were electrochemically polymerized without loss in redox activity of ferrocene group. Moreover, click chemistry was utilized in post-polymerization functionalization. For this purpose, three azide-containing polypyrroles, P(Py3N₃), P(Py4N₃), and P(Py6N₃) were electrochemically synthesized and subjected to click reaction in the presence of ethynylferrocene. CV studies of the post-polymerization functionalized polymers revealed quasi-reversible waves, while only P(Py6-post-Fc) showed the characteristic redox behavior of both polypyrrole and ferrocene. Thus, in this study preparation of a conducting homopolymers of pyrrole having covalently bonded ferrocene units was demonstrated and effect of spacer group is investigated.     

Synthesis and photoinduced transformation of helical aromatic polyamides containing atropisomeric biphenylene units and azobenzene segments in the main chain

Optically active helical polyamides were synthesized by condensation of axially dissymmetric (R)‐ or (S)‐6,6′‐diamino‐2,2′‐dimethylbiphenyl with aromatic dicarbonyl chlorides. The wholly aromatic polyamides obtained were soluble in various low‐polarity organic solvents such as tetrahydrofuran and chloroform, as well as in polar N,N‐dimethylacetamide. Excellent thermal stability of the helical structure was observed for the polyamide obtained with 4,4′‐dicarbonylbiphenyl chloride in refluxing N,N‐dimethylacetamide. Chiroptical data obtained from the circular dichroism spectra showed that the helical conformation of the polyamide containing azobenzene segments in the main chain can be transformed reversibly on irradiation with UV–visible light because of the trans–cis isomerization of the segments. Copyright © 2004 Society of Chemical Industry     

Transient N4-Acyl-DNA Protection against Cleavage by Restriction Endonucleases

A set of five N⁴-acyl-modified 2′-deoxycytidine 5′-triphosphates were incorporated into modified DNA by using phi29 DNA polymerase, and cleavage by selected restriction endonucleases was studied. Modified DNA containing N⁴-acyl functional groups in either one or both strands of a DNA molecule was resistant to the majority of restriction enzymes tested, whereas modifications outside of the recognition sequences were well tolerated. The N⁴-acylated cytidine derivatives were subjected to competitive nucleotide incorporation by using phi29 DNA polymerase, showing that a high-fidelity phi29 DNA polymerase efficiently used the modified analogues in the presence of its natural counterpart. These N⁴ modifications were also demonstrated to be easily removed in an aqueous ethanolamine solution, in which all steps, including primer extension, demodification, and cleavage by restriction endonuclease, could be performed in a one-pot procedure that eliminated additional purification stages. It is suggested that N⁴-modified nucleotides are promising building blocks for a programmable; transient; and, most importantly, straightforward DNA protection against specific endonucleases.     

Enzymatic Construction of Artificial Base Pairs: The Effect of Metal Shielding

Th formation of metal base pairs is a versatile method for the introduction of metal cations into nucleic acids that has been used in numerous applications including the construction of metal nanowires, development of energy, charge-transfer devices and expansion of the genetic alphabet. As an alternative, enzymatic construction of metal base pairs is an alluring strategy that grants access to longer sequences and offers the possibility of using such unnatural base pairs (UBPs) in SELEX experiments for the identification of functional nucleic acids. This method remains rather underexplored, and a better understanding of the key parameters in the design of efficient nucleotides is required. We have investigated the effect of methylation of the imidazole nucleoside ( dIm ^{n Me} TP ) on the efficiency of the enzymatic construction of metal base pairs. The presence of methyl substituents on dImTP facilitates the polymerase-driven formation of dIm^4Me −Ag^I− dIm and dIm^2MeTP −Cr^III− dIm base pairs. Steric factors rather than the basicity of the imidazole nucleobase appear to govern the enzymatic formation of such metal base pairs. We also demonstrate the compatibility of other metal cations rarely considered in the construction of artificial metal bases by enzymatic DNA synthesis under both primer extension reaction and PCR conditions. These findings open up new directions for the design of nucleotide analogues for the development of metal base pairs.     

Synthesis and characterization of novel photoactive polyamide derived from substituted fluorene by copper (I) catalyst

A novel polyamide has been successfully prepared through the reaction of 2,7‐dibromo‐9,9‐dioctylfluorene with 2,5‐dipiperazinedione in the presence of CuI, N,N′‐dimethylethylene diamine (DMEDA) and K₂CO₃ as base mixture and as catalyst. The structures of the monomer and the resulting model compound, as well as the structure, solution viscosity, solubility, molecular weights, thermal behavior, thermal stability, and light absorption and emission spectra of the resulting polyamide were characterized by means of FTIR, elemental analysis, ¹H‐NMR, ¹³C‐NMR, DSC, TGA, GPC and UV–visible absorption, and fluorescence emission spectrophotometers. The polyamide possesses excellent solubility in organic solvents such as tetrahydrofuran (THF), N‐methyl‐2‐pyrrolidone (NMP), N,N‐dimethylacetamide (DMAc), N,N‐dimethylformamide (DMF), ethylacetate, acetone, ethanol, pyridine, chloroform, and toluene at room temperature. The polyamide had inherent viscosity of 0.65 dL/g, and molecular weights of M_n= 4.25 × 10⁴ and M_w= 5.99 × 10⁴ g/mol. The polyamide had glass transition temperature (T_g) of 138°C, and 10% weight loss at 350°C in nitrogen. The polyamide showed strong UV absorption and blue emission in solution and in solid state. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Oligonucleotide synthesis onto poly(N‐acryloylmorpholine‐co‐N‐acryloxysuccinimide): Assessment of the resulting conjugates in a DNA sandwich hybridization test

A water‐soluble statistical poly(N‐acryloylmorpholine‐co‐N‐acryloxysuccinimide) [poly(NAM/NAS)] copolymer was studied for polymer–oligonucleotide (ODN) conjugate elaboration and for further use in diagnostic applications. Three different copolymers were first prepared by free‐radical solution polymerization with different N‐acryloylmorpholine (NAM) and N‐acryloxysuccinimide (NAS) molar ratios (80/20, 70/30, and 60/40). Their number‐average molecular weights ranged from 98,000 to 120,000 g/mol, as determined by aqueous size exclusion chromatography with an online light‐scattering detector. Then, polymer–ODN conjugates were obtained via a strategy consisting of the direct synthesis of ODNs onto polymer chains previously grafted onto a controlled pore glass support. Before the grafting of the polymer onto the solid support, a preliminary step was performed to bind a nucleotide starter along the polymer chain (via the reactive NAS units) to initiate automated DNA synthesis. To multiply the number of ODNs growing from starters, a branched phosphoramidite synthon [bearing two O‐dimethoxytrityl groups] was introduced at the first step of ODN elongation as a short sequence of four branched synthons alternated with three thymidine residues. Conjugates were assessed in a DNA sandwich hybridization test developed for hepatitis B virus detection. Sensitivity limits were evaluated and compared to those obtained with an other polymer, poly(maleic anhydride‐alt‐methyl vinyl ether) [poly(MA/MVE)]. A sensitivity limit of 2.6 × 10⁷ DNA copies/mL was reached with the poly(MA/MVE)–ODN conjugate at the capture phase and with the poly(NAM/NAS)–branched ODN conjugate at the detection phase of the test. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 3784–3795, 2004     

Polymeric coatings based on acrylic resin latexes from miniemulsion polymerization using hydrocarbon resins as osmotic agents

Waterborne acrylic resins with a solid content higher than 40 wt % were obtained by miniemulsion polymerization of methyl methacrylate, butyl acrylate, and acrylic acid using a hydrocarbon coumarone–indene resin (HCR) as osmotic agent. HCR is a cheap polymer widely used for coatings and pressure‐sensitive adhesives. The resin leads to a higher hydrophobicity for the acrylic latex film and acts as osmotic agent in miniemulsion polymerization preventing Ostwald ripening, leading to latexes with particle sizes, size distributions, and stability comparable to those obtained using n‐hexadecane as osmotic agent. However, the monomer conversion and molecular weight were lower, indicating the occurrence of a chain‐transfer reaction. Atomic force microscopy analysis demonstrated that a smooth film surface with phase‐separated morphology was formed when using HCR. Faster film hardness development was achieved with HCR comparing with hexadecane. Compared with market standard in a paint formulation, a similar performance was achieved. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40569.