5‐(Pyren‐1‐yl)uracil as a Base‐Discriminating Fluorescent Nucleobase in Pyrrolidinyl Peptide Nucleic Acids

A pyrene‐labeled uridine (U^Py) monomer for a pyrrolidinyl peptide nucleic acid with an alternating proline/2‐aminocyclopentanecarboxylic acid backbone (acpcPNA) was synthesized and incorporated into the PNA. The U^Py base in acpcPNA could specifically recognize the base A in its complementary DNA strand as determined by thermal denaturation (T_m) experiments. The fluorescence of the U^Py‐containing single‐stranded acpcPNA was very weak in aqueous buffer. In the presence of a complementary DNA target, the fluorescence was enhanced significantly (2.7–41.9 folds, depending on sequences). The fluorescence enhancement was specific to the pairing between U^Py and dA, making the U^Py‐modified acpcPNA useful as a hybridization‐responsive fluorescence probe for DNA‐sequence determination.     

Targeting of Single‐Stranded Oligonucleotides through Metal‐Induced Cyclization of Short Complementary Strands

A new strategy to cyclize short synthetic oligonucleotides on DNA or RNA target strands is described. The approach is based on metal‐templated cyclization of short synthetic oligonucleotides conjugated with two chelating 2,2′ : 6′,2′′‐terpyridine (Tpy) moieties at their 3′‐ and 5′‐ends. Cyclization after metal addition (Zn²⁺, Fe²⁺) was demonstrated by means of thermal‐denaturation experiments, MALDI‐Q‐TOF‐MS, and gel electrophoresis (PAGE). 1D‐ and 2D‐NMR Experiments were performed to analyze the association of complementary strands after metal‐mediated cyclization. Our protocol allows the efficient circularization of synthetic oligonucleotides. Thereby, the hybridization on a complementary strand was more efficient with an RNA target strand and a 2′‐O‐methylated circularized oligomer.     

Synthesis and Properties of Nonpolar DNA (Arylalkyl)phosphonates

The eight (arylalkyl)‐modified phosphoramidites (=(arylalkyl)phosphonamidites) 1 – 8 (Fig. 2) were synthesized (Schemes 1–3) and incorporated at different positions into 2′‐deoxyoligonucleotides. The [P(R)]‐ and [P(S)]‐diastereoisomers of the hexanucleotides 32 – 39 (Table 1) and of the dodecanucleotides 41 – 45 (Table 2) obtained were separated by means of reversed‐phase HPLC. UV, CD, and fluorescence spectroscopy were used to investigate the thermal stability (T_m) and the structural changes of their DNA duplexes with 5′‐d(CGCGCG)‐3′ and 5′‐d(ATGATTGACCTG)‐3′, respectively. The T_m values significantly depend on the place of modification (Table 2). A dangling‐end effect is observed when the [3‐(anthracen‐9‐yl)propyl]‐modified 8 is attached at the 5′‐terminus (see duplex with 45c ). In the case of the incorporation of aromatic moieties tethered via a methylene linker to the P‐atom (benzyl‐ and (naphthalen‐1‐ylmethyl)‐modified 1 and 6 , resp.), the duplexes with the [P(R)]‐oligonucleotides are more stable than those with the [P(S)]‐isomers, whereas in the case of longer alkyl chains at the P‐atom (see 2 – 5 ), the T_m values show the reverse tendency. The observed T_m differences are assigned to changes in base stacking (Figs. 6 and 7).     

Unwinding DNA and RNA with Synthetic Complexes: On the Way to Artificial Helicases

Synthetic helicases can be designed on the basis of ligands that bind more strongly to single‐stranded nucleic acids than to double‐stranded nucleic acids. This can be achieved with ligands containing phenyl groups, which intercalate into single strands, but due to their small size not into double strands. Moreover, two phenyl rings are combined with a distance that allows bis‐intercalation with only single strands and not double strands. In this respect, such ligands also mimic single‐strand binding (SSB) proteins. Exploration with more than 23 ligands, mostly newly synthesised, shows that the distance between the phenyl rings and between those and the linker influence the DNA unwinding efficiency, which can reach a melting point decrease of almost ΔT_m=50 °C at much lower concentrations than that with any other known artificial helicases. Conformational pre‐organisation of the ligand plays a decisive role in optimal efficiency. Substituents at the phenyl rings have a large effect, and increase, for example, in the order of H<F<Cl<Br, which illustrates the strong role of dispersive interactions in intercalation. Studies with homopolymers revealed significant selectivity: for example, with a ligand concentration of 40 μM at 35 °C, only GC double strands melt (ΔT_m=48 °C), whereas the AT strand remains untouched, and with poly(rA)–poly(rU) as an RNA model one observes unfolding at 29 °C with a concentration of only 30 μM .     

Anomeric DNA: Functionalization of α-d Anomers of 7-Deaza-2′-deoxyadenosine and 2′-Deoxyuridine with Clickable Side Chains and Click Adducts in Homochiral and Heterochiral Double Helices

Anomeric base pairs in heterochiral DNA with strands in the α-d and β-d configurations and homochiral DNA with both strands in α-d configuration were functionalized. The α-d anomers of 2′-deoxyuridine and 7-deaza-2′-deoxyadenosine were synthesized and functionalized with clickable octadiynyl side chains. Nucleosides were protected and converted to phosphoramidites. Solid-phase synthesis furnished 12-mer oligonucleotides, which were hybridized. Pyrene click adducts display fluorescence, a few of them with excimer emission. T_m values and thermodynamic data revealed the following order of duplex stability α/α-d ≫β/β-d ≥α/β-d . CD spectra disclosed that conformational changes occur during hybridization. Functionalized DNAs were modeled and energy minimized. Clickable side chains and bulky click adducts are well accommodated in the grooves of anomeric DNA. The investigation shows for the first time that anomeric DNAs can be functionalized in the same way as canonical DNA for potential applications in nucleic acid chemistry, chemical biology, and DNA material science.     

Synthesis,Base Pairing,and Fluorescence Properties of Oligonucleotides Containing 1H‐Pyrazolo[3,4‐d]pyrimidin‐6‐amine (8‐Aza‐7‐deazapurin‐2‐amine) as an Analogue of Purin‐2‐amine

The synthesis of the N⁹‐ and N⁸‐(β‐D ‐2′‐deoxyribonucleosides) 2 and 10 , respectively, of 8‐aza‐7‐deazapurin‐2‐amine (=1H‐pyrazolo[3,4‐d]pyrimidin‐6‐amine) is described. The fluorescence properties and the stability of the N‐glycosylic bond of 2 were determined and compared with those of the 2′‐deoxyribonucleosides 1 and 3 of purin‐2‐amine and 7‐deazapurin‐2‐amine respectively. From the nucleoside 2 , the phosphoramidite 14 was prepared, and oligonucleotides were synthesized. Duplexes containing compound 1 or 2 are slightly less stable than those containing 2′‐deoxyadenosine, while their CD spectra are rather different. The fluorescence of the nucleosides is strongly quenched (>95%) in single‐stranded as well as in duplex DNA. The residual fluorescence was used to determine the melting profiles, which gave T_m values similar to those determined from the UV melting curves.     

Temperature‐induced Molecular Chain Motions of Styrenic Triblock Copolymers Studied by Intrinsic Fluorescence Spectra

The temperature‐induced molecular chain motions of styrenic triblock copolymers (SBC), i.e. polystyrene‐block‐polybutadiene‐block‐polystyrene (SBS) and polystyrene‐block‐poly(ethylene‐co‐1‐butene)‐block‐polystyrene (SEBS), were studied by intrinsic fluorescence method. For SBS, the glass transition temperatures (T_gs) of B block and S block obtained by intrinsic fluorescence method were in good agreement with differential scanning calorimetry measurements (DSC). In the case of SEBS, an isoemission point was observed at about 310 nm at elevated temperatures, suggesting the slight conversion between the monomer and excimer emission. On this basis, the molecular chain motion of SEBS was monitored by both fluorescence intensity and excimer/monomer fluorescence ratio. Besides the T_gs of S block and EB blocks, a melting point (T_m) of weak crystalline in EB block was unambiguously determined by intrinsic fluorescence. Furthermore, it was found that the melting process directly led to the slight loosening of PS segments in interface and consequently the reduction of the amount of excimer. A reasonable mechanism was proposed to describe the molecular chain movements and phase transitions of SEBS upon heating. Moreover, the influence of temperature on the apparent activation energy of non‐radiative process (E_a^T) around T_g of S block was much stronger than that around T_g of B or EB blocks.     

A Nucleobase‐Discriminating Pyrrolo‐dC Click Adduct Designed for DNA Fluorescence Mismatch Sensing

New pyrrolo‐dC click adducts ( 4 and 5 ) tethered with a 1,2,3‐triazole skeleton were synthesized and oligonucleotides were prepared. The triazole system was either directly linked to the pyrrolo moiety ( 5 ) or connected via an n‐butyl linker ( 4 ). The quantum yield of nucleoside 5 (Φ=0.32), which is 10 times higher than those of 8‐methylpyrrolo‐dC ( 1 b , Φ=0.026) or the long linker derivative 4 (Φ=0.03), is maintained in oligonucleotides. Compound 5 was used as a nucleobase‐discriminating fluorescence sensor in duplex DNA. Excellent mismatch discrimination was observed when 5 was positioned opposite the four canonical nucleosides. Compound 5 has the potential to be used for SNP detection in long DNA targets when conventional techniques such as high resolution melt analysis fail.     

Reductive Coupling of Diynes at Rhodium Gives Fluorescent Rhodacyclopentadienes or Phosphorescent Rhodium 2,2’‐Biphenyl Complexes

Reactions of [Rh(κ²‐O,O‐acac)(PMe₃)₂] (acac=acetylacetonato) and α,ω‐bis(arylbutadiynyl)alkanes afford two isomeric types of MC₄ metallacycles with very different photophysical properties. As a result of a [2+2] reductive coupling at Rh, 2,5‐bis(arylethynyl)rhodacyclopentadienes ( A ) are formed, which display intense fluorescence (Φ=0.07–0.54, τ=0.2–2.5 ns) despite the presence of the heavy metal atom. Rhodium biphenyl complexes ( B ), which show exceptionally long‐lived (hundreds of μs) phosphorescence (Φ=0.01–0.33) at room temperature in solution, have been isolated as a second isomer originating from an unusual [4+2] cycloaddition reaction and a subsequent β‐H‐shift. We attribute the different photophysical properties of isomers A and B to a higher excited state density and a less stabilized T₁ state in the biphenyl complexes B , allowing for more efficient intersystem crossing S₁→T_n and T₁→S₀. Control of the isomer distribution is achieved by modification of the bis‐ (diyne) linker length, providing a fundamentally new route to access photoactive metal biphenyl compounds.     

Heterochiral DNA with Complementary Strands with α-d and β-d Configurations: Hydrogen-Bonded and Silver-Mediated Base Pairs with Impact of 7-Deazapurines Replacing Purines

Heterochiral DNA with hydrogen-bonded and silver-mediated base pairs have been constructed using complementary strands with nucleosides with α-d or β-d configuration. Anomeric phosphoramidites were employed to assemble the oligonucleotides. According to the T_m values and thermodynamic data, the duplex stability of the heterochiral duplexes was similar to that of homochiral DNA, but mismatch discrimination was better in heterochiral DNA. Replacement of purines by 7-deazapurines resulted in stable parallel duplexes, thereby confirming Watson–Crick-type base pairing. When cytosine was facing cytosine, thymine or adenine residues, duplex DNA formed silver-mediated base pairs in the presence of silver ions. Although the CD spectra of single strands with α-d configuration display mirror-like shapes to those with the β-d configuration, the CD spectra of the hydrogen-bonded duplexes and those with a limited number of silver pairs show a B-type double helix almost indistinguishable from natural DNA. Nonmelting silver ion–DNA complexes with entirely different CD spectra were generated when the number of silver ions was equal to the number of base pairs.     

Internal plasticization of poly(vinyl) chloride using glutamic acid as a branched linker to incorporate four plasticizers per anchor point

Internal plasticization of polyvinyl chloride (PVC) using thermal azide‐alkyne Huisgen dipolar cycloaddition between azidized PVC and electron‐poor acetylenediamides incorporating a branched glutamic acid linker resulted in incorporation of four plasticizing moieties per attachment point on the polymer chain. A systematic study incorporating either alkyl or polyethylene glycol esters provided materials with varying degrees of plasticization, with depressed T_g values ranging from ?1 °C to 62 °C. Three interesting trends were observed. First, T_g values of PVC bearing various internal plasticizers were shown to decrease with increasing chain length of the plasticizing ester. Second, branched internal plasticizers bearing triethylene glycol chains had lower T_g values compared to those with similar length long‐chain alkyl groups. Finally, thermogravimetric analysis of these internally plasticized PVC samples revealed that these branched internal plasticizers bearing alkyl chains are more thermally stable than similarity branched plasticizers bearing triethylene glycol units. These internal tetra‐plasticizers were synthesized and attached to PVC‐azide in three simple synthetic steps. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019 , 57, 1821–1835     

Oligonucleotides Containing Disaccharide Nucleosides

Disaccharide nucleosides with 2′‐O‐(D ‐arabinofuranosyl), 2′‐O‐(L ‐arabinofuranosyl), 2′‐O‐(D ‐ribopyranosyl), 2′‐O‐(D ‐erythrofuranosyl), and 2′‐O‐(5‐azido‐5‐deoxy‐D ‐ribofuranosyl) substituents were synthesized. These modified nucleosides were incorporated into oligonucleotides (see Table). Single substitution resulted in a ΔT_m of +0.5 to −1.4° for DNA/RNA and a ΔT_m of −0.8 to −4.7° for DNA/DNA duplexes. These disaccharide nucleosides can be well accommodated in RNA/DNA duplexes, and the presence of a NH₂−C(5″) group has a beneficial effect on duplex stability.     

Which Polyesters Can Mimic Polyethylene?

Self‐metathesis of erucic acid by [(PCy₃)(η‐C‐C₃H₄N₂Mes₂)Cl₂Ru = CHPh] (Grubbs second‐ generation catalyst) followed by catalytic hydrogenation and purification via the ester yields 1,26‐hexacosanedioate (>99% purity). Polyesterification with 1,26‐hexacosanediol, generated from the diester, affords polyester‐26,26, which features a T_m of 114 °C (T_c = 92 °C, ΔH_m = 160 J g⁻¹). Ultralong‐chain model polyesters‐38,23 (T_m = 109 °C) and −44,23 (T_m = 111 °C), generated via multistep procedures including acyclic diene metathesis polymerization, underline that melting points of such aliphatic polyesters do not gradually increase with methylene sequence chain length. Available data suggest that to mimic linear polyethylenes thermal properties, even longer sequences, amounting to at least four times a fatty acid chain, fully incorporated in a linear fashion are required.     

Synthesis and hybridization properties of 2′-O-(tetrazol-5-yl)ethyl-modified oligonucleotides

2′-O-(1H-Tetrazol-5-yl)ethyladenosine was synthesized using 2′-O-cyanoethyladenosine derivative as a key intermediate. The 2′-O-(1H-tetrazol-5-yl)ethyl modifications exhibited intriguing properties such as the change in the structure of the tetrazole residue between a protonated and a deprotonated form. The T_m experiments of various oligodeoxynucleotides having a 2′-O-(1H-tetrazol-5-yl)ethyl-modified adenosine showed reduced hybridization affinity in comparison to the unmodified oligonucleotides toward their complementary oligodeoxynucleotides. The mechanism of the reduced hybridization affinity was discussed on the basis of the structure and the physicochemical properties of the tetrazole moiety.     

Molecular Design for Reversing the Photoswitching Mode of Turning ON and OFF DNA Hybridization

A new photoswitch for DNA hybridization involving para‐substituted azobenzenes (such as isopropyl‐ or tert‐butyl‐substituted derivatives) with L ‐threoninol as a linker was synthesized. Irradiation of the modified DNA with visible light led to dissociation of the duplex owing to the destabilization effect of the bulky substituent on the trans‐azobenzene. In contrast, trans‐to‐cis isomerization (UV light irradiation) facilitated duplex formation. The direction of this photoswitching mode was entirely reversed relative to the previous system with an unmodified azobenzene on D ‐threoninol whose trans form turned on the hybridization, and cis form turned it off. Such reversed and reversible photoswitching of DNA hybridization was directly demonstrated by using fluorophore‐ and quencher‐attached oligonucleotides. Furthermore, it was revealed that the cis‐to‐trans thermal isomerization was greatly suppressed in the presence of the complementary strand owing to the formation of the more‐stable duplex in the cis form.     

Highly Sensitive Indicator‐Free Impedance Sensing of DNA Hybridization Based on Poly(m‐aminobenzenesulfonic acid)/TiO2 Nanosheet Membranes with Pulse Potentiostatic Method Preparation

A direct electrochemical detection procedure for DNA hybridization by using the electrochemical signal changes of conductive poly(m‐aminobenzenesulfonic) acid (PABSA)/TiO₂ nanosheet membranes, which were electropolymerized by using the pulse potentiostatic method, is reported. Due to the unique properties of TiO₂ nanoparticles, m‐aminobenzenesulfonic acid monomers tend to be adsorbed around the particles, and the electropolymerization efficiency is greatly improved. The combination of TiO₂ nanoparticles and PABSA resulted in a nanocomposite membrane with unique and novel nanosheet morphology that provides more activation sites and enhances the surface electron‐transfer rate. These characteristics were propitious for the magnification of PABSA electrochemical signals and the direct detection of DNA hybridization. Owing to the presence of abundant sulfonic acid groups, PABSA could overcome the drawbacks of polyaniline and be used to detect bioanalytes at physiological pH. DNA probes could be covalently attached to the sulfonic groups through the amines of DNA sequences by using an acyl chloride cross‐linking reaction. After immobilization of probe DNA, the electrochemical impedance value increased significantly compared to that of PABSA/TiO₂ nanosheet membranes, and then decreased dramatically after the hybridization reaction of the probe DNA with the complementary DNA sequence compared to that of the probe‐immobilized electrode. Electrochemical impedance spectroscopy was adopted for indicator‐free DNA biosensing, which had an eminent ability for the recognition between double‐base mismatched sequences or non‐complementary DNA sequences and complementary DNA sequences. A gene fragment, which is related to one of the screening genes for the transgenically modified plants, the cauliflower mosaic virus 35S gene was satisfactorily detected. This is the first report for the indicator‐free impedance DNA hybridization detection by using PABSA/TiO₂ membranes under neutral conditions.     

Thermal Stability of Modified i‐Motif Oligonucleotides with Naphthalimide Intercalating Nucleic Acids

In continuation of our investigation of characteristics and thermodynamic properties of the i‐motif 5′‐d[(CCCTAA)₃CCCT)] upon insertion of intercalating nucleotides into the cytosine‐rich oligonucleotide, this article evaluates the stabilities of i‐motif oligonucleotides upon insertion of naphthalimide (1H‐benzo[de]isoquinoline‐1,3(2H)‐dione) as the intercalating nucleic acid. The stabilities of i‐motif structures with inserted naphthalimide intercalating nucleotides were studied using UV melting temperatures (T_m) and circular dichroism spectra at different pH values and conditions (crowding and non‐crowding). This study indicated a positive effect of the naphthalimide intercalating nucleotides on the stabilities of the i‐motif structures compared to the wild‐type structure which is in contrast to a previous observation for a pyrene‐intercalating nucleotide showing a decrease in T_m values.     

Temperature response of aqueous solutions of pyrene end‐labeled poly(N‐isopropylacrylamide)s probed by steady‐state and time‐resolved fluorescence

Aqueous solutions of a series of monodisperse poly(N‐isopropylacrylamide)s end‐labeled with n‐butyl‐1‐pyrene at one or both chain ends (Py_n‐PNIPAMs with n = 1 or 2) were studied by turbidimetry, light scattering, and fluorescence. For a given polymer concentration and heating rate, the cloud point (T_c) of an aqueous Py_n‐PNIPAM solution, determined by turbidimetry, was found to increase with the number‐average molecular weight (M_n) of the polymer. The steady‐state fluorescence spectra and time‐resolved fluorescence decays of Py_n‐PNIPAM aqueous solutions were analyzed and all parameters retrieved from these analyses were found to be affected as the solution temperature passed through T_c, the solution cloud point, and T_m, the temperature where dehydration of PNIPAM occurred. The trends obtained by fluorescence to characterize the aqueous Py_n‐PNIPAM solutions as a function of temperature were found to be consistent with the model proposed for telechelic PNIPAM by Koga et al. in 2006. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 308–318     

Direct selective detection of genomic DNA from coliform using a fiber optic biosensor

Fiber optic biosensors operated in a total internal reflection format were prepared based on covalent immobilization of 25mer lacZ single-stranded nucleic acid probe. Genomic DNA from Escherichia coli was extracted and then sheared by sonication to prepare fragments of approximately 300mer length. Other targets included a 25mer fully complementary lacZ sequence, 100mer polymerase chain reaction (PCR) products containing the lacZ sequence at various locations, and non-complementary DNA including genomic samples from salmon sperm. Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases. The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution. The presence of high concentrations of non-complementary genomic DNA had little effect on extent or speed of hybridization of complementary oligonucleotides. Detection of genomic fragments containing the lacZ sequence was possible in as little as 20 s by observation of the steady-state fluorescence intensity increase or by time-dependent rate of fluorescence intensity changes.     

Influence of monofunctional reactants on the physical properties of dimer acid‐based polyamides

Dimer acid‐based polyamides were synthesized by condensation polymerization in the absence and presence of monofunctional reactants. Acetic acid, oleic acid and propyl amine were used as monofunctional reactants. The influences of the equivalent percentage (E%) and type of monofunctional reactant on the physical properties of dimer acid‐based polyamides such as glass transition temperature (T_g), melting point (T_m), heat of fusion (ΔH), degree of polymerization (DP), number average molecular weight (M_n), and kinematic viscosity were investigated. The molecular weight and viscosity of dimer acid‐based polyamides decreased with the increase in equivalent percentage of monofunctional reactant. Differential scanning calorimetry (DSC) studies showed that acetic acid and propyl amine had higher effect on the thermal properties of polyamides than that of oleic acid. In the case of polyamides prepared in the presence of acetic acid, the values of T_g, T_m, and ΔH of the polyamides increased remarkably with the increase in acetic acid content. On the contrary, propyl amine had a decreasing effect on the values of T_g, T_m, and ΔH of the polyamides. Incorporation of oleic acid into the polymer structure had no significant effect on the values of T_g and T_m of the dimer acid‐based polyamides. Copyright © 2006 John Wiley & Sons, Ltd.