Nucleoside and oligonucleotide pyrene conjugates with 1,2,3-triazolyl or ethynyl linkers: synthesis,duplex stability,and fluorescence changes generated by the DNA-dye connector

Fluorescent nucleosides and oligonucleotides functionalized with pyrene were synthesized using ‘click’ chemistry or the Sonogashira cross-coupling reaction. The dye was connected to position-7 of 7-deaza-2′-deoxyguanosine or to the 2′-deoxyribofuranose moiety. Four different DNA-dye connectors with 1,2,3-triazolyl residues or triple bonds were constructed. Phosphoramidites of the pyrene conjugates (9, 14, 25) were prepared and used in solid-phase synthesis. Short linkers (2, 4) destabilize DNA, while long linkers (1) increased duplex stability. Nucleosides and oligonucleotides with single dye incorporations show linker dependent fluorescence. Linker dependent excimer emission with pyrenes in proximal positions was also observed. A ‘superchromophore’ formed by the 7-deaza-2′-deoxyguanosine ethynylpyrene conjugate shows strong red shifted fluorescence emission at 495 nm.     

Advances in nucleic acid architectures for electrochemical sensing

Nucleic acid–based electrochemical sensors are ideally suited to the detection of molecular targets for which enzymatic detection or direct electrochemical oxidation – reduction reactions are not possible. Moreover, the versatility of nucleic acids in their ability to bind a great variety of target types, from small molecules to single-entity mesoscopic targets, makes them attractive receptors for the development of electrochemical biosensors. In this brief opinion piece, we discuss field advances from the past two years. We hope the works highlighted here will inspire the community to pursue creative designs enabling the detection of larger and more complex targets with a specific focus on analytical validation and translation into preclinical or clinical applications.     

Discovery of Novel Sultone Fused Berberine Derivatives as Promising Tdp1 Inhibitors

A new type of berberine derivatives was obtained by the reaction of berberrubine with aliphatic sulfonyl chlorides. The new polycyclic compounds have a sultone ring condensed to C and D rings of a protoberberine core. The reaction conditions were developed to facilitate the formation of sultones with high yields without by-product formation. Thus, it was shown that the order of addition of reagents affects the composition of the reaction products: when sulfochlorides are added to berberrubine, their corresponding 9-O-sulfonates are predominantly formed; when berberrubine is added to pre-generated sulfenes, sultones are the only products. The reaction was shown to proceed stereo-selectively and the cycle configuration was confirmed by 2D NMR spectroscopy. The inhibitory activity of the synthesized sultones and their 12-brominated analogs against the DNA-repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1), an important target for a potential antitumor therapy, was studied. All derivatives were active in the micromolar and submicromolar range, in contrast to the acyclic analogs and 9-O-sulfonates, which were inactive. The significance of the sultone cycle and bromine substituent in binding with the enzyme was confirmed using molecular modeling. The active inhibitors are mostly non-toxic to the HeLa cancer cell line, and several ligands show synergy with topotecan, a topoisomerase 1 poison in clinical use. Thus, novel berberine derivatives can be considered as candidates for adjuvant therapy against cancer.     

Modeling DNA beacons at the mesoscopic scale

We report model calculations on DNA single strands which describe the equilibrium dynamics and kinetics of hairpin formation and melting. Modeling is at the level of single bases. Strand rigidity is described in terms of simple polymer models; alternative calculations performed using the freely rotating chain and the discrete Kratky-Porod models are reported. Stem formation is modeled according to the Peyrard-Bishop-Dauxois Hamiltonian. The kinetics of opening and closing is described in terms of a diffusion-controlled motion in an effective free-energy landscape. Melting profiles, dependence of melting temperature on loop length, and kinetic time scales are in semiquantitative agreement with experimental data obtained from fluorescent DNA beacons forming poly(T) loops. Variation in strand rigidity is not sufficient to account for the large activation enthalpy of closing and the strong loop length dependence observed in hairpins forming poly(A) loops. Implications for modeling single strands of DNA or RNA are discussed.     

Quantitative analysis of oxidative DNA damage induced by high-voltage pulsed discharge with cavitation

Pulsed discharge is used for sterilization and disinfection, but the details of the molecular mechanisms remain largely unknown. Since pulsed discharge generates reactive oxygen species (ROS), we analyzed the oxidative DNA damages after pulsed discharge treatment to consider the involvement of ROS in the damaging process. We applied pulsed discharge with cavitation to plasmid DNA molecules and estimated the yields of the damages by agarose gel electrophoresis. The treated DNA contained various oxidative DNA damages, including single and double strand breaks and base lesions. The yields of the damages increased in response to the energy used for pulsed discharge. We also measured the yield of 8-hydroxyguanine (8-OH-G), one of the major oxidative base lesions, in the treated plasmid DNA by mass spectrometry quantitatively and found that the yield of the oxidative base lesion corresponded to the increment of the applied energy. In addition, we observed the involvement of mutM gene, which is responsible for repair of 8-OH-G, in the increased sensitivity of Escherichia coli to pulsed discharge. Therefore, ROS seem to mediate the sterilization ability of pulsed discharge.     

1,2,3-三氮唑基团连接的双丙烯酸酯单体环聚合形成PEG聚合物刷的合成及表征

通过带有PEG官能团的双丙烯酸酯大分子单体的RAFT环聚合反应合成含有十一元环重复结构的PEG大分子刷．不同PEG长度的连接1,2,3-三氮唑的双丙烯酸酯大分子单体通过点击化学反应合成．PEG侧链的较大位阻效应影响双丙烯酸酯大分子单体的聚合行为,以致于双丙烯酸酯大分子单体优先进行环化聚合反应而不发生交联反应．核磁数据和凝胶渗透色谱证明高效的环化聚合反应,而且没有副反应发生．PEG大分子刷在紫外光激发下有较强的荧光,而荧光则强烈依赖于聚合物刷的浓度,这归因于环聚合物在水中的聚集．PEG大分子刷的荧光能被DNA淬灭．     

Constructions for Terraces and R‐Sequencings,Including a Proof That Bailey's Conjecture Holds for Abelian Groups

Every abelian group of even order with a noncyclic Sylow 2‐subgroup is known to be R‐sequenceable except possibly when the Sylow 2‐subgroup has order 8. We construct an R‐sequencing for many groups with elementary abelian Sylow 2‐subgroups of order 8 and use this to show that all such groups of order other than 8 also have terraces. This completes the proof of Bailey's Conjecture in the abelian case: all abelian groups other than the noncyclic elementary abelian 2‐groups have terraces. For odd orders it is known that abelian groups are R‐sequenceable except possibly those with noncyclic Sylow 3‐subgroups. We show how the theory of narcissistic terraces can be exploited to find R‐sequencings for many such groups, including infinitely many groups with each possible of Sylow 3‐subgroup type of exponent at most 3¹² and all groups whose Sylow 3‐subgroups are of the form or .     

Coverage percentage and coverage rate of different DNA nanostructures grown on a mica substrate

In this study, we simultaneously grew different DNA nanostructures, including 5 helix ribbon (5HR), double-crossover (DX), and double-crossover with single hairpin (DX-SH) lattices, on a mica substrate, and we analyzed the concentration-dependent coverage percentages of 5HR at a fixed concentration of DX ([DX]), DX at a fixed [5HR], and DX-SH with both fixed [5HR] and [DX]. The structural features of the various nanostructures formed through mica-assisted growth annealing were confirmed via atomic force microscopy. To assess the temperature-dependent coverage rates on the given substrates, we introduced two simple models, i.e., the equipartition model (EPM) and the un-equipartition model (UEPM) which provide information on the amount of coverage for each of the given temperature. EPM provides an equal amount of the coverage percentage per temperature change due to its linear nature, but UEPM does not since it has a non-linear nature. The coverage percentages obtained by these models allow us to calculate, as a function of temperature, the coverage rates of 5HR at a fixed [DX] and DX-SH for both fixed [5HR] and [DX]. Although with EPM the coverage rates for the 5HR and DX-SH decreased linearly as the temperature decreased, they decreased non-linearly as the temperature decreased with UEPM.     

A Supercharged Fluorescent Protein as a Versatile Probe for Homogeneous DNA Detection and Methylation Analysis

Supercharged proteins are a new class of functional proteins with exceptional stability and potent ability to deliver bio‐macromolecules into cells. As a proof‐of‐principle, a novel application of supercharged proteins as a versatile biosensing platform for nucleic acid detection and epigenetics analysis is presented. Taking supercharged green fluorescent protein (ScGFP) as the signal reporter, a simple turn‐on homogenous method for DNA detection has been developed based on the polyionic nanoscale complex of ScGFP/DNA and toehold strand displacement. This assay shows high sensitivity and potent ability to detect single‐base mismatch. Furthermore, combined with bisulfite conversion, this ScGFP‐based assay was further applied to analyze site‐specific DNA methylation status of genomic DNA extracted from real human colon carcinoma tissue sample with ultrahigh sensitivity (4 amol methylated DNA).