Solid-phase synthesis of bleomycin group antibiotics. Construction of a 108-member deglycobleomycin library

The bleomycins (BLMs) are structurally related glycopeptide antibiotics isolated from Streptomyces verticillus that mediate the sequence-selective oxidative damage of DNA and RNA. Deglycobleomycin, which lacks the carbohydrate moiety, cleaves DNA analogously to bleomycin itself, albeit less potently, and has been used successfully for analyzing the functional domains of bleomycin. Although structural modifications to bleomycin and deglycobleomycin have been reported, no bleomycin or deglycobleomycin analogue having enhanced DNA cleavage activity has yet been described. The successful synthesis of a deglycobleomycin on a solid support has permitted the facile solid-phase synthesis of 108 unique deglycobleomycin analogues through parallel solid-phase synthesis. Each of the deglycobleomycin analogues was synthesized efficiently; the purity of each crude product was greater than 60%, as determined by HPLC integration. The solid-phase synthesis of the deglycobleomycin library provided near-milligram to milligram quantities of each deglycobleomycin, thereby permitting characterization by (1)H NMR and high-resolution mass spectrometry. Each analogue demonstrated supercoiled plasmid DNA relaxation above background cleavage; the library included two analogues that mediated plasmid relaxation to a greater extent than the parent deglycobleomycin molecule.     

Solid-phase synthesis of bleomycin group antibiotics. Elaboration of deglycobleomycin A(5)

The solid-phase syntheses of two deglycobleomycin A(5) analogues were achieved using a commercially available polystyrene resin containing triphenylmethyl-linked spermidine. The final products were deblocked and released from the resin, analyzed, and purified by C(18) reversed phase HPLC and characterized by high-field (1)H NMR spectroscopy and mass spectrometry. The purified products relaxed supercoiled plasmid DNA in a concentration-dependent fashion and to the same extent as authentic material derived from natural BLM A(5).     

Conformationally constrained analogues of bleomycin A5

The bleomycin (BLM) group antitumor antibiotics are glycopeptide-derived natural products shown to cause sequence selective lesions in DNA. Prior studies have indicated that the linker region, composed of the methylvalerate and threonine residues, may be responsible for a conformational bend in the agent required for efficient DNA cleavage. We have synthesized a number of conformationally constrained methylvalerate analogues and incorporated them into deglycobleomycin A(5) congeners using our recently reported procedure for the solid phase construction of (deglyco)bleomycin and its analogues. These analogues were designed to probe the effects of conformational constraint of the native valerate moiety. Initial experiments indicated that the constrained molecules, none of which mimic the conformation proposed for the natural valerate linker, possessed DNA cleavage activity, albeit with potencies less than that of (deglyco)BLM and lacking sequence selectivity. Further experiments demonstrated that these analogues failed to produce alkali-labile lesions in DNA or sequence selective oxidative damage in RNA. However, two of the conformationally constrained deglycoBLM analogues were shown to mediate RNA cleavage in the absence of added Fe(2+). The ability of the analogues to mediate the oxygenation of small molecules was also assayed, and it was shown that they were as competent in the transfer of oxygen to low molecular weight substrates as the parent compound.     

High-density DNA alignment on an Au(111) surface starting from folded DNA

High-density uniform DNA alignment on a metal substrate is essential for creating sensitive DNA devices. We develop a self-sensing DNA alignment process starting from folded DNA to achieve high-density, uniform DNA alignment on an Au(111) surface. We demonstrate that folded DNA plays a critical role in avoiding DNA aggregation and distributing the DNA uniformly on an Au(111) surface at the greatest density and quality ever attained. We also verify that the distributed, folded DNA can be stimulated to align only when the appropriate buffer flow is applied. This selective self-sensing DNA alignment on an Au surface will be a key technology for creating dynamic DNA sensors and switches.     

Immobilization of DNA onto poly(dimethylsiloxane) surfaces and application to a microelectrochemical enzyme-amplified DNA hybridization assay

This paper describes immobilization of DNA onto the interior walls of poly(dimethylsiloxane) (PDMS) microsystems and its application to an enzyme-amplified electrochemical DNA assay. DNA immobilization was carried out by silanization of the PDMS surface with 3-mercaptopropyltrimethoxysilane to yield a thiol-terminated surface. 5'-acrylamide-modified DNA reacts with the pendant thiol groups to yield DNA-modified PDMS. Surface-immobilized DNA oligos serve as capture probes for target DNA. Biotin-labeled target DNA hybridizes to the PDMS-immobilized capture DNA, and subsequent introduction of alkaline phosphatase (AP) conjugated to streptavidin results in attachment of the enzyme to hybridized DNA. Electrochemical detection of DNA hybridization benefits from enzyme amplification. Specifically, AP converts electroinactive p-aminophenyl phosphate to electroactive p-aminophenol, which is detected using an indium tin oxide interdigitated array (IDA) electrode. The IDA electrode eliminates the need for a reference electrode and provides a steady-state current that is related to the concentration of hybridized DNA. At present, the limit of detection of the DNA target is 1 nM in a volume of 20 nL, which corresponds to 20 attomoles of DNA.     

Hybridization of oligonucleotide by using DNA self-assembled monolayer

The interaction between DNA immobilized on surface and oligonucleotides at the interface is important in detection and diagnostic processes. However, it is difficult to immobilize DNA with maintaining its activity and to realize an efficient hybridization in previous methods. Here, to establish a novel DNA-functionalized surface, the DNA self-assembled monolayer (SAM) was constructed on a gold substrate using thiolated DNA composed of double-stranded (ds) and single-stranded (ss) portion. The DNA SAM was characterized by surface plasmon resonance (SPR), XPS. The hybridization of ss portion of DNA was attempted using the SAM, and in situ monitored by SPR. XPS measurement indicated that the thiolated DNA could form a stable monolayer on a gold substrate through sulfur–gold interaction. SPR measurement implied that the long axis of the DNA standing on the substrate. These results indicated formation of the DNA SAM on the substrate. Hybridization of target DNA containing a complementary sequence for the probe portion was observed by SPR. Moreover, one mismatch of oligonucleotide could be distinguished using the DNA SAM. The SPR result indicates that hybridization of target DNA and probe DNA on the DNA SAM occurs on the DNA SAM.     

A novel strategy to construct a flat-lying DNA monolayer on a mica surface

Flat-lying, densely packed DNA monolayers in which DNA chains are well organized have been successfully constructed on a mica surface by dropping a droplet of a DNA solution on a freshly cleaved mica surface and subsequently transferring the mica to ultrapure water for developing. The formation kinetics of such monolayers was studied by tapping mode atomic force microscopy (TMAFM) technique. A series of TMAFM images of DNA films obtained at various developing times show that before the sample was immersed into water for developing the DNA chains always seriously aggregated by contacting, crossing, or overlapping and formed large-scale networks on the mica surface. During developing, the fibers of DNA networks gradually dispersed into many smaller fibers up to single DNA chains. At the same time, the fibers or DNA chains also experienced rearrangement to decrease electrostatic repulsion and interfacial Gibbs free energy. Finally, a flat-lying, densely packed DNA monolayer was formed. A formation mechanism of the DNA monolayers was proposed that consists of aggregation, dispersion, and rearrangement. The effects of both DNA and Mg2+ concentration in the formation solution on DNA monolayer formation were also investigated in detail.     

DNA折纸术研究进展

DNA折纸术是近年来提出的一种全新的DNA自组装的方法,是DNA纳米技术与DNA自组装领域的一个重大进展。与传统的DNA自组装技术不同,DNA折纸术通过将一条长的DNA单链（通常为基因组DNA）与一系列经过设计的短DNA片段进行碱基互补,能够可控地构造出高度复杂的纳米图案或结构,在新兴的纳米领域中具有广泛的潜在应用。本文在介绍DNA折纸术相关原理的基础上,就DNA折纸术的起源、发展及其在DNA芯片、纳米元件与材料等领域的潜在应用进行了概述,探讨了DNA折纸术未来可能的发展方向。     

Development of 3D printed mesofluidic devices to elute and concentrate DNA

To understand structural variation for personal genomics, an extensive ensemble of large DNA molecules will be required to span large structural variations. Nanocoding, a whole‐genome analysis platform, can analyze large DNA molecules for the construction of physical restriction maps of entire genomes. However, handling of large DNA is difficult and a system is needed to concentrate large DNA molecules, while keeping the molecules intact. Insert technology was developed to protect large DNA molecules during routine cell lysis and molecular biology techniques. However, eluting and concentrating DNA molecules has been difficult in the past. Utilizing 3D printed mesofluidic device, a proof of principle system was developed to elute and concentrate lambda DNA molecules at the interface between a solution and a poly‐acrylamide roadblock. The matrix allowed buffer solution to move through the pores in the matrix; however, it slowed down the progression of DNA in the matrix, since the molecules were so large and the pore size was small. Using fluorescence intensity of the insert, 84% of DNA was eluted from the insert and 45% of DNA was recovered in solution from the eluted DNA. DNA recovered was digested with a restriction enzyme to determine that the DNA molecules remained full length during the elution and concentration of DNA.     

High resolution mass spectrometry based profiling of diet-related deoxyribonucleic acid adducts

Exposure of DNA to endo- and exogenous DNA binding chemicals can result in the formation of DNA adducts and is believed to be the first step in chemically induced carcinogenesis. DNA adductomics is a relatively new field of research which studies the formation of known and unknown DNA adducts in DNA due to exposure to genotoxic chemicals. In this study, a new UHPLC-HRMS(/MS)-based DNA adduct detection method was developed and validated. Four targeted DNA adducts, which all have been linked to dietary genotoxicity, were included in the described method; O⁶-methylguanine (O⁶-MeG), O⁶-carboxymethylguanine (O⁶-CMG), pyrimidopurinone (M1G) and methylhydroxypropanoguanine (CroG). As a supplementary tool for DNA adductomics, a DNA adduct database, which currently contains 123 different diet-related DNA adducts, was constructed. By means of the newly developed method and database, all 4 targeted DNA adducts and 32 untargeted DNA adducts could be detected in different DNA samples. The obtained results clearly demonstrate the merit of the described method for both targeted and untargeted DNA adduct detection in vitro and in vivo, whilst the diet-related DNA adduct database can distinctly facilitate data interpretation.     

DNA end resection and its role in DNA replication and DSB repair choice in mammalian cells

DNA end resection has a key role in double-strand break repair and DNA replication. Defective DNA end resection can cause malfunctions in DNA repair and replication, leading to greater genomic instability. DNA end resection is initiated by MRN-CtIP generating short, 3′-single-stranded DNA (ssDNA). This newly generated ssDNA is further elongated by multiple nucleases and DNA helicases, such as EXO1, DNA2, and BLM. Effective DNA end resection is essential for error-free homologous recombination DNA repair, the degradation of incorrectly replicated DNA and double-strand break repair choice. Because of its importance in DNA repair, DNA end resection is strictly regulated. Numerous mechanisms have been reported to regulate the initiation, extension, and termination of DNA end resection. Here, we review the general process of DNA end resection and its role in DNA replication and repair pathway choice.Subject terms: Double-strand DNA breaks, Cell signalling     

Manipulation of globular DNA molecules for sizing and separation

Handling large DNA molecules, such as chromosomal DNA, has become necessary due to recent developments in genome science. However, large DNA molecules are fragile and easily broken by shear stress accompanying flow in solution. This fragility causes difficulties in the preparation and handling of large DNA molecules. This study demonstrates the transition of DNA from a coiled to a globular form, which is highly condensed. This state suppresses DNA fragmentation due to shear stress in solution. The transition enables large DNA molecules to undergo mechanical manipulation. We confirmed that the fluorescence intensity of stained globular DNA increases with increasing length, suggesting that the resistance of globular DNA to shear stress is the factor that allows analysis of large DNA by flow cytometry.     

Charge Transfer through the DNA Base Stack

Whether the DNA base pair stack might serve as a medium for efficient, long-range charge transfer has been debated almost since the first proposal of the double-helical structure of DNA. The consequences of long-range radical migration through DNA are important with respect to understanding carcinogenesis and mutagenesis. Double-helical DNA has in its core a stacked array of aromatic heterocyclic base pairs, and this molecular π stack represents a unique system in which to explore the chemistry of electron transfer. We designed a family of metal complexes which bind to DNA by intercalative stacking within the helix; these metallointercalators may be usefully applied in probing DNA-mediated electron transfer. Here we describe a range of electron transfer reactions we carried out which are mediated by the DNA base paired stack. In some cases, DNA serves as a bridge, and spectroscopic analyses permit us to probe how the π stack couples DNA-bound donors and acceptors. These studies point to the sensitivity of coupling to DNA intercalation. However, if the DNA π stack effectively bridges donors and acceptors, the base-pair stack itself might serve not only as a conduit for electron transfer in DNA, but also in reactions initiated from a remote position. We carried out a series of reactions involving oxidative damage to DNA arising from the remotely positioned oxidant on the helix. The implications of long-range charge migration through DNA to effect damage are substantial. As in other DNA-mediated charge transfers, these reactions are highly dependent on DNA intercalation and the integrity of the intervening base-pair stack, but not on molecular distance. Furthermore, a physiologically important DNA lesion, the thymine dimers, can be reversed in a reaction initiated by electron transfer. This repair reaction can also be promoted from a distance as a result of long-range charge migration through the DNA base pair stack.     

Sequence-specific detection of femtomolar DNA via a chronocoulometric DNA sensor (CDS): effects of nanoparticle-mediated amplification and nanoscale control of DNA assembly at electrodes

We herein report a novel nanoparticle-based electrochemical DNA detection approach. This DNA sensor is based on a "sandwich" detection strategy, which involves capture probe DNA immobilized on gold electrodes and reporter probe DNA labeled with gold nanoparticles that flank the target DNA sequence. Electrochemical signals are generated by chronocoulometric interrogation of [Ru(NH(3))(6)](3+) that quantitatively binds to surface-confined capture probe DNA via electrostatic interactions. We demonstrated that the incorporation of a gold nanoparticle in this sensor design significantly enhanced the sensitivity and the selectivity. Nanoscale control of the self-assembly process of DNA probes at gold electrodes further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar (zeptomoles) DNA targets and exhibited excellent selectivity against even a single-base mismatch. In addition, this novel DNA sensor showed fairly good reproducibility, stability, and reusability.     

Separation of single-stranded DNA fragments at a 10-nucleotide resolution by stretching in microfluidic channels

We report a novel DNA separation method by tethering DNA chains to a solid surface and then stretching the DNA chains with an electric field. The anchor is such designed that the critical force to detach a DNA chain is independent of its size. Because the stretching force is proportional to the DNA net charge, a gradual increase of the electric field leads to size-based removal of the DNA strands from the surface and thus DNA separation. Here we show that this method, originally proposed for separation of long double-stranded DNA chains (>10,000 base pairs), is also applicable to single-stranded (ss) DNA fragments with less than 100 nucleotides (nt). Theoretical analysis indicates that the separation resolution is limited by the fluctuation forces on tethered DNA chains. By employing a microfluidic platform with narrow channels filled with a buffer of low ionic conductivity, we are able to apply a strong electric field to the DNA fragments with negligible Joule heating. Upon stepwise increments of the electric field, we demonstrate efficient separation of short ssDNA fragments at a 10-nt resolution.     

寡聚酰胺为探针的电化学DNA生物传感器

DNA分子中的碱基对可以长程传递电荷, DNA分子中的碱基π堆积结构为电荷的长程传递提供了良好的通道. 电荷在DNA分子中的传递受碱基序列的影响, 利用这种性质可以构建DNA碱基错配检测的电化学传感器. 寡聚酰胺能和DNA以小沟绑定方式高亲和力地结合, 并且具有序列识别功能, 本文以带有硝基官能团的寡聚酰胺分子为电化学探针, 设计了电化学DNA生物传感器. 结果显示, 寡聚酰胺与DNA修饰电极作用后, 电化学响应显著增强, 并且可以作为检测DNA碱基错配的电化学探针分子.     

Gaussian fluctuations in tethered DNA chains

In a recent work [Gao et al., Appl. Phys. Lett. 134, 113902 (2007)], we reported a novel DNA separation method by tethering DNA chains to a solid surface and then stretching the DNA chains with an electric field. The anchor is such designed that the critical force to detach a DNA chain is independent of its length. Because the stretching force is proportional to the DNA net charge, a gradual increase of the electric field leads to size-based removal of the DNA strands from the surface and thus DNA separation. Originally proposed for separation of long double-stranded DNA chains (>10 000 bps), this method has been proven useful also for short single-stranded DNA fragments (<100 bases) for which the fluctuation force induced by the solvent becomes significant. Here we show that the fluctuation force can be approximately represented by a gaussian model for tethered DNA chains. Analytical expressions have been derived to account for the dependence of the fluctuation force on the surface confinement, the polymer chain length, and the DNA tethering potential. The theoretical predictions are found to coincide with experiment.     

Effects of bridge ions, DNA species, and developing temperature on flat-lying DNA monolayers

Recently, we have successfully constructed flat-lying DNA monolayers on a mica surface (J. Phys. Chem. B 2006, 110, 10792-10798). In this work, the effects of various factors including bridge ions, DNA species, and developing temperature on the configuration of DNA monolayers have been investigated by atomic force microscopy (AFM) in detail. AFM results show that the species of bridge ions and developing temperature play a crucial role during the formation process. For example, the divalent cation Zn2+ resulted in many DNA chains stuck side by side in the monolayers due to the strong interactions between it and DNA's bases or the mica surface. Most DNA chain's conglutinations disappeared when the developing temperature was higher than 40 degrees C. Cd2+ and Ca2+ produced more compact DNA monolayers with some obvious aggregations, especially for the DNA monolayers constructed by using Ca2+ as the bridge ion. Co2+ produced well-ordered, flat-lying DNA monolayers similar to that of Mg2+. Furthermore, it was found that the flat-lying DNA monolayers could still form on a mica surface when plasmid DNA pBR 322 and linear DNA pBR 322/Pst I were used as the DNA source. Whereas, it was hard to form DNA monolayers on a (3-aminopropyl)triethoxysilane-mica surface because the strong interactions between DNA and substrate prevented the lateral movement of DNA molecules. These results suggested that the appropriate interactions between divalent cations and DNA or mica surface were important for the formation of flat-lying DNA monolayers. The obtained information is a necessary supplement to our previous studies on the formation kinetics of such monolayers and may be useful for practical application of the monolayers and further theoretical studies.     

Complexation induced by weak interaction between DNA and PEO-b-P4VP below the CMC of the polymer

The complexation between circular DNA and individual chains of PEO-b-P4VP with a relatively long PEO block and a short P4VP block is highly controllable when the interaction between DNA and the polymer is weak enough. When one circular DNA chain is taken into consideration, and the polymer concentration is far below its critical micelle concentration(CMC), polymer chains are absorbed by DNA chain due to the interaction between the negatively charged DNA chain and the slightly positively charged P4VP block chains. After the adsorption/complexation, the DNA chain is converted into a nanoring(type 1). In the nanoring, the DNA chain is sufficiently wrapped by the polymer and adopts a fully stretched conformation, so that the DNA compact ratio in the nanorings is close to 1. When the polymer concentration is close to but lower than the CMC, the free polymer chains in the solution are adsorbed not only by the DNA chain but also by the polymer chains that have already been adsorbed on the DNA chain. As a result, the circular DNA chain adsorbs more polymer chains, and thus the resultant nanoring(type 2) has a larger width. In the type 2 nanoring, the DNA chain is slightly compressed; the DNA compact ratio is only about 2-3. Therefore, complexation induced by the weak interaction between DNA and PEO-b-P4VP below the CMC can produce narrow-disperse and large nanorings with a perimeter of micrometers, which are difficult to prepare by existing methods.