pH‐Independent Recognition of the dG ⋅ dC Base Pair in Triplex DNA: 9‐Deazaguanine N7‐(2′‐Deoxyribonucleoside) and Halogenated Derivatives Replacing Protonated dC

Triplex-forming oligonucleotides (TFOs) containing 9-deazaguanine N⁷-(2′-deoxyribonucleoside) 1a and halogenated derivatives 1b,c were synthesized employing solid-phase oligonucleotide synthesis. For that purpose, the phosphoramidite building blocks 5a – c and 8a – c were synthesized. Multiple incorporations of 1a – c in place of dC were performed within TFOs, which involved the sequence of five consecutive 1a – c ⋅ dG ⋅ dC triplets as well as of three alternating 1a – c ⋅ dG ⋅ dC and dT ⋅ dA ⋅ dT triplets. These TFOs were designed to bind in a parallel orientation to the target duplex. Triplex forming properties of these oligonucleotides containing 1a – c in the presence of Na⁺ and Mg²⁺ were studied by UV/melting-curve analysis and confirmed by circular-dichroism (CD) spectroscopy. The oligonucleotides containing 1a in the place of dC formed stable triplexes at physiological pH in the case of sequence of five consecutive 1a ⋅ dG ⋅ dC triplets as well as three alternating 1a – c ⋅ dG ⋅ dC and dT ⋅ dA ⋅ dT triplets. The replacement of 1a by 9-halogenated derivatives 1b,c further enhanced the stability of DNA triplexes. Nucleosides 1a – c also stabilized duplex DNA.     

Regioselective arylation of 2′-deoxyribonucleosides on amido or imino sites by copper(II)-mediated direct coupling with arylboronic acids

N¹-Aryl derivatives of 2′-deoxyguanosine (dG) were synthesized by copper(II)-mediated coupling of dG with arylboronic acids. Analogous aryl derivatives of 2′-deoxyinosine (dIn), 2′-deoxyuridine (dU), thymidine (T), 2′-deoxyadenosine (dA), and 2′-deoxycytidine (dC) were also conveniently synthesized by this method. Arylation took place preferentially on the amido functions in dG and dIn and the imino functions in dU or T. Remarkably, the nucleosides themselves served as internal ligands as well as reactants.     

四甲基吡啶卟啉与核酸相互作用的稳态和时间分辨光谱

采用稳态吸收和荧光光谱、圆二色谱和皮秒时间分辨荧光光谱手段, 研究了5,10,15,20-四[4-(N-甲基吡啶)]卟啉(TMPyP4)与腺嘌呤(A)、鸟嘌呤(G)、胸腺嘧啶(T)和胞嘧啶(C)等4种碱基, 以及相应的核苷、核苷酸和单链DNA的结合能力和光谱学性质. 研究结果发现, 嘌呤与TMPyP4的结合能力比嘧啶的强. 对于某一碱基系列, 结合能力强弱顺序依次为: 碱基~核苷<核苷酸<单链DNA. 时间分辨荧光谱研究发现, 除鸟嘌呤外, 核酸和TMPyP4复合物的荧光动力学均含有快(1~2 ns)和慢(约10 ns)两个衰减过程, 它们分别是由激基复合体和环境极性对激发态TMPyP4分子的影响所致. 单链DNA能诱导TMPyP4产生诱导圆二色信号, 而单分子(碱基、核苷、核苷酸)则无此功能.     

DNA fragment conformations. I—methods for the assignment of DNA fragment proton resonances. 1H NMR spectra of dApTpGpT and dApCpApTpGpT

A general method for the assignment of DNA fragment proton resonances, especially for the sugar protons, has been presented and used to interpret the 400 MHz proton spectra of dApTpGpT and dApCpApTpGpT in neutral aqueous solution. Only fine splittings of about 3 Hz are observed in the H-2″ resonances, and the total splitting is larger for the H-2′ (≈29 Hz) than for the H-2″ (22–23 Hz) multiplets. The purine and pyrimidine resonances can be distinguished on the basis of the H-2″ and H-2″ chemical shifts. The resonances of the H-2′ and H-2″ protons (above and below the sugar plane, respectively) of dA and dG exhibit chemical shifts of 2.65—2.80 ppm, while those of dC and dT residues are located at higher fields between 1.95 and 2.40 ppm. At high temperature (≥60°C), δH-2′>YδH-2″ for the purine family, while δH-2′ « δH-2″ in the case of the pyrimidine family. Except for the terminal residue, the H-3′ resonances of dA and dG are located at lower fields compared with those of the dC and dT residues. The same is true for the H-4′ resonances. In general δA_1′>δG_1′ and in the case of self complementary duplexes the H-1′ and H-2′ chemical shift variations versus temperature are found to be larger for the dC than for the dT residues.     

Naphthalene Imide Conjugates: Formation of Supramolecular Assemblies,and the Encapsulation and Release of Dyes through DNA‐Mediated Disassembly

We report the synthesis of two new amphiphilic conjugates 1 and 2 based on naphthalene di‐ and monoimide chromophores and the investigation of their photophysical, self‐assembly and DNA‐binding properties. These conjugates showed aqueous good solubility and exhibited strong interactions with DNA and polynucleotides such as poly(dG?dC)–poly(dG?dC) and poly(dA?dT)–poly(dA?dT). The interaction of these conjugates with DNA was evaluated by photo‐ and biophysical techniques. These studies revealed that the conjugates interact with DNA through intercalation with association constants in the order of 5–8×10⁴ M ^?1. Of these two conjugates, bolaamphiphile 1 exhibited a supramolecular assembly that formed vesicles with an approximate diameter of 220 nm in the aqueous medium at a critical aggregation concentration of 0.4 mM , which was confirmed by SEM and TEM. These vesicular structures showed a strong affinity for hydrophobic molecules such as Nile red through encapsulation. Uniquely, when exposed to DNA the vesicles disassembled, and therefore this transformation could be utilised for the encapsulation and release of hydrophobic molecules by employing DNA as a stimulus.     

DNA-Triggered Enhancement of Singlet Oxygen Production by Pyridinium Alkynylanthracenes

There is an ongoing interest in ¹O₂ sensitizers, whose activity is selectively controlled by their interaction with DNA. To this end, we synthesized three isomeric pyridinium alkynylanthracenes 2 o – p and a water-soluble trapping reagent for ¹O₂. In water and in the absence of DNA, these dyes show a poor efficiency to sensitize the photooxygenation of the trapping reagent as they decompose due to electron transfer processes. In contrast, in the presence of DNA ¹O₂ is generated from the excited DNA-bound ligand. The interactions of 2 o – p with DNA were investigated by thermal DNA melting studies, UV/vis and fluorescence spectroscopy, and linear and circular dichroism spectroscopy. Our studies revealed an intercalative binding with an orientation of the long pyridyl-alkynyl axis parallel to the main axis of the DNA base pairs. In the presence of poly(dA : dT), all three isomers show an enhanced formation of singlet oxygen, as indicated by the reaction of the latter with the trapping reagent. With green light irradiation of isomer 2 o in poly(dA : dT), the conversion rate of the trapping reagent is enhanced by a factor >10. The formation of ¹O₂ was confirmed by control experiments under anaerobic conditions, in deuterated solvents, or by addition of ¹O₂ quenchers. When bound to poly(dG : dC), the opposite effect was observed only for isomers 2 o and 2 m , namely the trapping reagent reacted significantly slower. Overall, we showed that pyridinium alkynylanthracenes are very useful intercalators, that exhibit an enhanced photochemical ¹O₂ generation in the DNA-bound state.     

Thermodynamic profiles of the DNA binding of benzophenanthridines sanguinarine and ethidium: A comparative study with sequence specific polynucleotides

Energetics of the binding of two known classical DNA intercalating molecules, ethidium and sanguinarine with four sequence specific polynucleotides, poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dT).poly(dA-dT), and poly(dA).poly(dT) have been compared under identical conditions. The binding of both the molecules was characterized by strong stabilization of the polynucleotides against thermal strand separation in optical melting as well as differential scanning calorimetry studies. Isothermal titration calorimetry results revealed that the binding of both sanguinarine and ethidium to poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), and poly(dG).poly(dC) was exothermic and favoured by negative enthalpy changes. On the other hand, the binding of both molecules to poly(dA).poly(dT) was endothermic and entropy driven. The binding affinity values obtained from isothermal titration calorimetry data was in close proximity to that derived from thermal melting data. The heat capacity changes obtained from temperature dependence of the enthalpy change gave negative values in the range (?0.4 to 1.25) kJ · mol^?1 · K^?1 for the binding of ethidium and sanguinarine to these polynucleotides. The variations in the values indicate important differences in the formation of the complexes. New insights into the energetics and specificity aspects of interaction of these molecules to DNA have emerged from these studies.     

Theoretical study of the interaction of tetramethylammonium with double-stranded oligonucleotides

Computations are performed on the interaction specificities of tetramethylammonium (TMA) for double-stranded oligonucleotides held in the B conformation. The effects of base sequence and chain length are investigated. In the short oligomers (helices formed from dinucleoside monophosphates and trinucleoside diphosphates), the interaction energies of TMA are larger in the major groove of (dG)_n · (dC)_n than in the minor groove of either (dA)_n · (dT)_n or (dA—dT)_n. Upon lengthening the oligomers, and owing to the gradual shaping of the grooves of the helix and cumulative effect of the phosphates, TMA is shown to increasingly favor the minor groove of (dA)_n · (dT)_n with respect to the major groove of (dG)_n · (dC)_n, with a sizeable energy difference computed at the pentanucleoside hexaphosphate level. The binding of TMA in the minor groove of (dA)_n · (dT)_n involves stabilizing contacts with several sites, on the bases and on the deoxyriboses. Configurations locating the cation closer to the thymine strand are slightly preferred over configurations locating it closer to the adenine strand.     

Calorimetric and thermal analysis studies on the binding of phenothiazinium dye thionine with DNA polynucleotides

Binding of the phenothaizinium dye thionine with four sequence specific deoxyribopolynucleotides, poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dT).poly(dA-dT), and poly(dA).poly(dT) has been investigated by means of thermal helix melting, isothermal titration calorimetry, and differential scanning calorimetry experiments. The binding affinity values evaluated from isothermal titration calorimetry suggests that thionine exhibits the highest binding affinity to poly(dG-dC).poly(dG-dC). The binding to poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), and poly(dG).poly(dC) is exothermic and favoured by negative enthalpy changes while binding to poly(dA).poly(dT) is endothermic and anomalous. The values of heat capacity changes of the interaction are negative and in the range (?0.4 to ?0.5) kJ · K^?1 · mol^?1. The binding is characterized by strong stabilization of the polynucleotides against thermal strand separation. The binding affinity values derived from thermal melting data are in excellent agreement with those obtained from isothermal titration calorimetry data. Insights into the energetic aspects and guanine–cytosine selectivity of the DNA interaction of thionine have been obtained from these studies.     

EFFICIENCY OF PHOTOAFFINITY LABELING DNA HOMOPOLYMERS AND COPOLYMERS WITH ETHIDIUM MONOAZIDE *

Abstract— Photoaffinity labeling of synthetic DN As with ethidium monoazide was studied to determine if the efficiency of adduct formation was related to DNA sequence. Equilibrium drug binding to DNA homopolymers and copolymers was quanitified by phase partition techniques. The amount of drug bound to a deoxypolymer at equilibrium was then compared to the fraction of ethidium analog covalently-linked following photoactivation at the same drug/DNA input ratio. There were significant sequence-related differences in the ability of the photoaffinity probe to label DNA covalently. The efficiency of covalent-adduct formation decreased in the order poly(dG-dC)^. poly(dG-dC)> poly-(dG). poly(dC)poly(dA-dT). poly(dA-dT)poly(dA). poly(dT). Ethidium monoazide was about 2-fold more efficient in labeling deoxyhomopolymers and deoxycopolymers composed of G-C pairs than the A-T base counterparts. In low ionic buffers (0.015 M Na⁺), the efficiency of photoactivation decreased with increasing ethidium monoazide concentrations. However. the base sequence effect was observed over a 40-fold range of drug concentrations. Therefore, the amount of ethidium monoazide bound to a DNA site after irradiation does not appear to represent the true affinity of the drug for that site.     

Estimation of gas-phase acidities of deoxyribonucleosides: An experimental and theoretical study

We determined the gas-phase acidities (ΔH_acid) of four deoxyribonucleosides, i.e., 2′-deoxyadenosine (dA), 2′-deoxyguanosine (dG), 2′-deoxycytidine (dC), and 2′-deoxythymidine (dT) by applying the extended kinetic method. The negatively charged proton-bound hetero-dimeric anions, [A-H-B]⁻ of the deoxyribonucleosides (A) and reference compounds (B) were generated under electrospray ionization conditions. Collision-induced dissociation spectra of [A-H-B]⁻ were recorded at four different collision energies using a triple quadrupole mass spectrometer. The abundance ratios of the individual monomeric product ions were used to determine the ΔH_acid of the deoxyribonucleosides. The obtained ΔH_acid value follows the order dA7>dC7>dT7>dG. The ΔG_acid (298 K) values were determined by using ΔG_acid=ΔH_acid-TΔS_acid where the ΔH_acid and ΔS_acid values were determined directly from the kinetic method plots. The ΔH_acid values were also predicted for the deoxyribonucleosides at the B3LYP/6-311+G**//B3LYP/6-311G** level of theory. The acidity trend obtained from the computational investigation shows good agreement with that obtained experimentally by the extended kinetic method. Theoretical calculations provided the most preferred deprotonation site as C5′-OH from sugar moiety in case of dA, and as −NH₂ (dC and dG) or -NH- (dT) from nitrogenous base moiety in the case of other deoxyribonucleosides.     

Kinetics of end-to-end collision in short single-stranded nucleic acids

A novel fluorescence-based method, which entails contact quenching of the long-lived fluorescent state of 2,3-diazabicyclo[2.2.2]-oct-2-ene (DBO), was employed to measure the kinetics of end-to-end collision in short single-stranded oligodeoxyribonucleotides of the type 5'-DBO-(X)n-dG with X = dA, dC, dT, or dU and n = 2 or 4. The fluorophore was covalently attached to the 5' end and dG was introduced as an efficient intrinsic quencher at the 3' terminus. The end-to-end collision rates, which can be directly related to the efficiency of intramolecular fluorescence quenching, ranged from 0.1 to 9.0 x 10(6) s(-1). They were strongly dependent on the strand length, the base sequence, as well as the temperature. Oligonucleotides containing dA in the backbone displayed much slower collision rates and significantly higher positive activation energies than strands composed of pyrimidine bases, suggesting a higher intrinsic rigidity of oligoadenylate. Comparison of the measured collision rates in short single-stranded oligodeoxyribonucleotides with the previously reported kinetics of hairpin formation indicates that the intramolecular collision is significantly faster than the nucleation step of hairpin closing. This is consistent with the configurational diffusion model suggested by Ansari et al. (Ansari, A.; Kuznetsov, S. V.; Shen, Y. Proc.Natl. Acad. Sci. USA 2001, 98, 7771-7776), in which the formation of misfolded loops is thought to slow hairpin formation.     

Effects of Bases on the Stabilities of Nucleoside C4'''' Radicals

C4'-H bond dissociation enthalpies of nucleosides were predicted using theoretical methods to a precision of 1-2 Kcal/mol. It was found that the stability of the C4' nucleoside radical is slightly dependent on the base. The orders of stability are dA < dG < dT < dC for deoxynucleosides and U < G < A = C for nucleosides.     

Photoswitchable Formation of a DNA Interstrand Cross‐Link by a Coumarin‐Modified Nucleotide

A coumarin‐modified pyrimidine nucleoside ( 1 ) has been synthesized using a Cu^I‐catalyzed click reaction and incorporated into oligodeoxynucleotides (ODNs). Interstrand cross‐links are produced upon irradiation of ODNs containing 1 at 350 nm. Cross‐linking occurs through a [2+2] cycloaddition reaction with the opposing thymidine, 2′‐deoxycytidine, or 2′‐deoxyadenosine. A much higher reactivity was observed with dT than dC or dA. Irradiation of the dT‐ 1 and dC‐ 1 cross‐linked products at 254 nm leads to a reversible ring‐opening reaction, while such phenomena were not observed with dA‐ 1 adducts. The reversible reaction is ultrafast and complete within 50–90 s. Consistent photoswitching behavior was observed over 6 cycles of irradiation at 350 nm and 254 nm. To the best of our knowledge, this is the first example of photoswitchable interstrand cross‐linking formation induced by a modified pyrimidine nucleoside.     

Sequence-specific control of azobenzene assemblies by molecular recognition of DNA

We investigated the molecular recognition between the amphiphile AzoAde, which is composed of azobenzene in the hydrophobic and adenine in the hydrophilic portion of the molecule, and oligonucleotides having a homogeneous base (dA30, dT30, dG30, and dC30) at the air-water interface. On the basis of the complementary base-pairing of DNA in the duplex, orderly arrangement of AzoAde on templated dT30 was examined using pi-A isotherm, UV-vis RAS, FT-IR RAS, and XPS measurements. Although there was little interaction between AzoAde and mismatched oligonucleotides (dA30, dG30, and dC30), AzoAde prepared on a dT30 subphase stoichiometrically assembled and interacted with dT30, subsequently forming a J-form assembly at the air-water interface. AFM observation of the LB films revealed the nanostructure of the J-formed AzoAde monolayer on the dT30 subphase as well as the domain structures of the H-formed monolayers on the other oligonucleotide subphases. Therefore, dT30 has a potential application as a template for assembling AzoAde at the air-water interface.     

Synthesis and Characterization of a Water‐Soluble Carboxylated Polyfluorene and Its Fluorescence Quenching by Cationic Quenchers and Proteins

We have developed a new intermediate monomer, 2,7‐[bis(4,4,5,5‐tetramethyl‐1,3,2‐dioxaborolan‐2‐yl)‐9,9‐bis(3‐(tert‐butyl propanoate))]fluorene, that allows the easy synthesis of water‐soluble carboxylated polyfluorenes. As an example, poly[9,9′‐bis(3′′‐propanoate)fluoren‐2,7‐yl] sodium salt was synthesized by the Suzuki coupling reaction, and the properties of the polymer were studied in aqueous solutions of different pH. Fluorescence quenching of the polymer by different cationic quenchers (MV²⁺, MV⁴⁺, and NO₂MV²⁺; MV=methyl viologen) was studied, and the quenching constants were found to be dependent on the charge and electron affinity of the quencher molecule and the pH of the medium. The largest quenching constant was observed to be 1.39×10⁸ M ^?1 for NO₂MV²⁺ at pH 7. The change in polymer fluorescence upon interaction with different proteins was also studied. Strong fluorescence quenching of the polymer was observed in the presence of cytochrome c, whereas weak quenching was observed in the presence of myoglobin and bovine serum albumin. Lysozyme quenched the polymer emission at low protein concentrations, and the quenching became saturated at high protein concentrations. Under similar experimental conditions, the polymer showed improved quenching efficiencies toward cationic quenchers and a more selective response to proteins relative to other carboxylated conjugated polymers.     

Synthesis and nucleic acid-binding properties of water-soluble porphyrins appending platinum(II) complexes.

We synthesized two water-soluble porphyrins appending platinum(II) complexes [alpha,beta-(4a) and alpha,alpha-(4b) 5,15-bis(2-trans-[PtCl(NH3)2]N-2-aminoethylaminocarbonylphenyl) 2,3,7,8,12,13,17,18-octamethylporphyrin] and studied their reactions with a variety of nucleic acids [disodium adenosine-5'-monophosphate (AMP), disodium guanosine-5'-monophosphate (GMP), disodium thymidine-5'-monophosphate (TMP), disodium cytidine-5'-monophosphate (CMP), synthetic polymer poly(dG)-poly(dC), poly(dA)-poly(dT)] by 1H-NMR, UV-vis and FAB-MS spectroscopies. Based on the denaturation experiments of synthetic nucleic acid polymers, we conclude that the presence of the porphyrins (5.6 microM) does not cause significant changes in the melting temperature of poly(dA)-poly(dT) (28 microM) (deltaT=1 degrees C) and shows reannealing. On the other hand, gradual melting of poly(dG)-poly(dC) (28 microM) occurs at a low temperature (deltaT= -27 degrees C) in the presence of the porphyrins (5.6 microM), and the solutions do not show reannealing phenomena. The results of UV-vis and 1H-NMR experiments revealed that the porphyrins bind to guanine bases and that the porphyrins bind to GMP more strongly than to the other nucleotides. The binding modes between the porphyrins and synthetic nucleic acids are affected more by the coordination of the nucleobase [poly(dG)-poly(dC)] to the Pt(II) in the porphyrins than by Coulomb and hydrophobic interactions.     

Synthesis and incorporation of a furan-modified adenosine building block for DNA interstrand crosslinking

2'-O-(3-(Furan-2-yl)propyl)adenosine was synthesized and evaluated for interstrand crosslink (ICL) formation in DNA duplexes. In situ oxidation of the furan moiety with NIS showed rapid crosslink formation to dA and dC, while dT and dG were inactive.