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Rapid identification of DNA oxidative damage sites is of great significance for disease diagnosis. In this work, electric field-regulated click reaction surface-enhanced Raman spectroscopy (e-Click-SERS) was developed aiming at the rapid and specific analysis of furfural, the biomarker of oxidative damage to the 5-carbon site of DNA deoxyribose. In e-Click-SERS, cysteamine-modified porous Ag filaments (cys@p-Ag) were prepared and used as electrodes, amine-aldehyde click reaction sites, and SERS substrates. Cysteamine was controlled as an "end-on" conformation by setting the voltage of cys@p-Ag at -0.1 V, which ensures its activity in participating in the amine-aldehyde click reaction during the detection of furfural. Benefiting from this, the proposed e-Click-SERS method was found to be sensitive, rapid-responding, and interference-resistant in analyzing furfural from plasma. The method detection limits of furfural were 5 ng mL -1 in plasma, and the whole "extraction and detection" procedure was completed within 30 min with satisfactory recovery. Interference from 13 kinds of common plasma metabolites was investigated and found to not interfere with the analysis, according to the exclusive adaptation of the amine-aldehyde click reaction. Notably, the e-Click-SERS technique allows in situ analysis of biological samples, which offers great potential to be a point-of-care testing tool for detecting DNA oxidative damage.

In this work, we studied the fragmentation dynamics of 2-deoxy-d-ribose (DR) in solution that arises from the double ionization of a water molecule in its primary hydration shell. This process was modeled in the framework of ab initio molecular dynamics. The charge unbalanced in the solvent molecules produces a Coulomb explosion with the consequent release of protons with kinetic energy in the few electronvolts range, which collide with the surrounding molecules in solution inducing further chemical reactions. In particular, we observe proton collisions with the solute molecule DR, which leads to a complete ring opening. In DNA, damage to the DR moiety may lead to DNA strand breaking. This mechanism can be understood as one of the possible steps in the radiation-induced fragmentation of DNA chains. [ABSTRACT FROM AUTHOR]

Formalin-fixed paraffin-embedded (FFPE) tissues are rarely used for screening DNA adducts of carcinogens because the harsh conditions required to reverse the formaldehyde-mediated DNA cross-links can destroy DNA adducts. We recently adapted a commercial silica-based column kit used in genomics to manually isolate DNA under mild conditions from FFPE tissues of rodents and humans and successfully measured DNA adducts of several carcinogens including aristolochic acid I (AA-I), 4-aminobiphenyl (4-ABP), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (Yun et al. (2013) Anal. Chem. 85, 4251-8, and Guo et al. (2016) Anal. Chem. 88, 4780-7). The DNA retrieval methodology is robust; however, the procedure is time-consuming and labor intensive, and not amenable to rapid throughput processing. In this study, we have employed the Promega Maxwell 16 MDx system, which is commonly used in large scale genomics studies, for the rapid throughput extraction of DNA. This system streamlines the DNA isolation procedure and increases the sample processing rate by about 8-fold over the manual method (32 samples versus 4 samples processed per hour). High purity DNA is obtained in satisfactory yield for the measurements of DNA adducts by ultra performance liquid chromatography-electrospray-ionization-ion trap-multistage scan mass spectrometry. The measurements show that the levels of DNA adducts of AA-I, 4-ABP, and PhIP in FFPE rodent and human tissues are comparable to those levels measured in DNA from matching tissues isolated by the commercial silica-based column kits and in DNA from fresh frozen tissues isolated by the conventional phenol-chloroform extraction method. The isolation of DNA from tissues is one major bottleneck in the analysis of DNA adducts. This rapid throughput methodology greatly decreases the time required to process DNA and can be employed in large-scale epidemiology studies designed to assess the role of chemical exposures and DNA adducts in cancer risk. [ABSTRACT FROM AUTHOR]

DNA unwinding plays a major role in many biological processes, such as replication, transcription, and repair. It can lead to local melting and strand separation and can serve as a key mechanism to promote access to the separate strands of a double-stranded DNA. While DNA unwinding has been investigated extensively by DNA cyclization and single-molecule studies on a length-scale of kilo base pairs, it is neither fully understood at the base pair level nor at the level of molecular interactions. By employing a torque acting on the termini of DNA oligonucleotides during molecular dynamics free energy simulations, we locally unwind the central part of a DNA beyond an elastic (harmonic) regime. The simulations reproduce experimental results on the twist elasticity in the harmonic regime (characterized by a mostly quadratic free energy change with respect to changes in twist) and a deformation up to 7° was found as a limit of the harmonic response. Beyond this limit the free energy increase per twist change dropped dramatically coupled to local base pair disruptions and significant deformation of the nucleic acid backbone structure. Restriction of the DNA bending flexibility resulted in a stiffer harmonic response and an earlier onset of the anharmonic response. Whereas local melting with a complete disruption of base pairing and flipping of nucleotides was observed in case of an AT rich central segment strong backbone changes and changes in the stacking arrangements were observed in case of a GC rich segment. Unrestrained MD simulations starting from locally melted DNA reformed regular B-DNA after 50-300 ns simulation time. The simulations may have important implications for understanding DNA recognition processes coupled with significant structural alterations. [ABSTRACT FROM AUTHOR]

The rapidly developing field of bionanotechnology requires detailed knowledge of the mechanisms of interaction between inorganic matter and biomolecules. Under conditions different from those in an aqueous solution, however, the chemistry of these systems is elusive and may differ dramatically from their interactions in vitro and in vivo. Here, we report for the first time a photoemission study of a metal silver-DNA interface, formed in vacuo, in comparison with DNA-Ag+ and fluorescent DNA-Ag complexes formed in solution. The high-resolution photoelectron spectra reveal that in vacuo silver atoms interact mainly with oxygen atoms of the phosphodiester bond and deoxyribose in DNA, in contrast to the behavior of silver ions, which interact preferentially with the nitrogen atoms of the bases. This offers new insight into the mechanism of DNA metallization, which is of importance in creating metal-bio interfaces for nanotechnology applications. [ABSTRACT FROM AUTHOR]

A polarizable environment, prominently the solvent, responds to electronic changes in biomolecules rapidly. The knowledge of conformational relaxation of the biomolecule itself, however, may be scarce or missing. In this work, we describe in detail the structural changes in DNA undergoing electron transfer between two adjacent nucleobases. We employ an approach based on averaging of tens to hundreds of thousands of nonequilibrium trajectories generated with molecular dynamics simulation, and a reduction of dimensionality suitable for DNA. We show that the conformational response of the DNA proceeds along a single collective coordinate that represents the relative orientation of two consecutive base pairs, namely, a combination of helical parameters shift and tilt. The structure of DNA relaxes on time scales reaching nanoseconds, contributing marginally to the relaxation of energies, which is dominated by the modes of motion of the aqueous solvent. The concept of reaction path averaging (RPA), conveniently exploited in this context, makes it possible to filter out any undesirable noise from the nonequilibrium data, and is applicable to any chemical process in general. [ABSTRACT FROM AUTHOR]

The novel evolutionary engineering method ExSELEX (genetic alphabet expansion for systematic evolution of ligands by exponential enrichment) provides high-affinity DNA aptamers that specifically bind to target molecules, by introducing an artificial hydrophobic base analogue as a fifth component into DNA aptamers. Here, we present a newer version of ExSELEX, using a library with completely randomized sequences consisting of five components: four natural bases and one unnatural hydrophobic base, 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). In contrast to the limited number of Ds-containing sequence combinations in our previous library, the increased complexity of the new randomized library could improve the success rates of high-affinity aptamer generation. To this end, we developed a sequencing method for each clone in the enriched library after several rounds of selection. Using the improved library, we generated a Ds-containing DNA aptamer targeting von Willebrand factor A1-domain (vWF) with significantly higher affinity (KD = 75 pM), relative to those generated by the initial version of ExSELEX, as well as that of the known DNA aptamer consisting of only the natural bases. In addition, the Ds-containing DNA aptamer was stabilized by introducing a mini-hairpin DNA resistant to nucleases, without any loss of affinity (KD = 61 pM). This new version is expected to consistently produce high-affinity DNA aptamers. [ABSTRACT FROM AUTHOR]

We describe an unprecedented DNA-catalyzed DNA cleavage process in which a radical-based reaction pathway cleanly results in excision of most atoms of a specific guanosine nucleoside. Two new deoxyribozymes (DNA enzymes) were identified by in vitro selection from N40 or N100 random pools initially seeking amide bond hydrolysis, although they both cleave simple single-stranded DNA oligonucleotides. Each deoxyribozyme generates both superoxide (O2-• or HOO•) and hydrogen peroxide (H2O2) and leads to the same set of products (3'-phosphoglycolate, 5'-phosphate, and base propenal) as formed by the natural product bleomycin, with product assignments by mass spectrometry and colorimetric assay. We infer the same mechanistic pathway, involving formation of the C4' radical of the guanosine nucleoside that is subsequently excised. Consistent with a radical pathway, glutathione fully suppresses catalysis. Conversely, adding either superoxide or H2O2 from the outset strongly enhances catalysis. The mechanism of generation and involvement of superoxide and H2O2 by the deoxyribozymes is not yet defined. The deoxyribozymes do not require redox-active metal ions and function with a combination of Zn2+ and Mg2+, although including Mn2+ increases the activity, and Mn2+ alone also supports catalysis. In contrast to all of these observations, unrelated DNA-catalyzed radical DNA cleavage reactions require redox-active metals and lead to mixtures of products. This study reports an intriguing example of a well-defined, DNA-catalyzed, radical reaction process that cleaves single-stranded DNA and requires only redox-inactive metal ions. [ABSTRACT FROM AUTHOR]

(-)-Lomaiviticin A (1) is a C2-symmetric cytotoxin that contains two diazofluorene functional groups and which induces double-strand breaks (DSBs) in DNA. Evidence suggests DNA cleavage is initiated by hydrogen atom abstraction from the deoxyribose backbone. Here we demonstrate the formation of the vinyl radicals 1· and 2· from 1 by 1,7-addition of thio to the diazofluorenes. These radicals can affect hydrogen atom abstraction from methanol and acetone. The first addition of thiol to 1 proceeds at a much greater rate than the second. The diazosulfide 5 formed en route to 1· has been detected at -50 °C and undergoes decomposition to 1· with a half-life of 110 min at -20 °C under air. These data, which constitute the first direct evidence for the generation of 1· and 2· from 1, provide insights into the mechanism of DNA cleavage by 1. [ABSTRACT FROM AUTHOR]

Cationic liposomes (CLs) are widely studied as carriers of DNA and short-interfering RNA for gene delivery and silencing, and related clinical trials are ongoing. Optimization of transfection efficiency (TE) requires understanding of CL-nucleic acid nanoparticle (NP) interactions with cells, NP endosomal pathways, endosomal escape, and events leading to release of active nucleic acid from the lipid carrier. Here, we studied endosomal pathways and TE of surface-functionalized CL-DNA NPs in PC-3 prostate cancer cells displaying overexpressed integrin and neuropilin-1 receptors. The NPs contained RGD-PEG-lipid or RPARPAR-PEG-lipid, targeting integrin, and neuropilin-1 receptors, respectively, or control PEG-lipid. Fluorescence colocalization using Rab11-GFP and Lysotracker enabled simultaneous colocalization of NPs with recycling endosome (Rab11) and late endosome/lysosome (Rab7/Lysotracker) pathways at increasing mole fractions of pentavalent MVL5 (+5 e) at low (10 mol %), high (50 mol %), and very high (70 mol %) membrane charge density (σM). For these cationic NPs (lipid/DNA molar charge ratio, ρchg = 5), the influence of membrane charge density on pathway selection and transfection efficiency is similar for both peptide-PEG NPs, although, quantitatively, the effect is larger for RGD-PEG compared to RPARPAR-PEG NPs. At low σM, peptide-PEG NPs show preference for the recycling endosome over the late endosome/lysosome pathway. Increases in σM, from low to high, lead to decreases in colocalization with recycling endosomes and simultaneous increases in colocalization with the late endosome/lysosome pathway. Combining colocalization and functional TE data at low and high σM shows that higher TE correlates with a larger fraction of NPs colocalized with the late endosome/lysosome pathway while lower TE correlates with a larger fraction of NPs colocalized with the Rab11 recycling pathway. The findings lead to a hypothesis that increases in σM, leading to enhanced late endosome/lysosome pathway selection and higher TE, result from increased nonspecific electrostatic attractions between NPs and endosome luminal membranes, and conversely, enhanced recycling pathway for NPs and lower TE are due to weaker attractions. Surprisingly, at very high σM, the inverse relation between the two pathways observed at low and high σM breaks down, pointing to a more complex NP pathway behavior. [ABSTRACT FROM AUTHOR]

The motors that drive double-stranded DNA (dsDNA) genomes into viral capsids are among the strongest of all biological motors for which forces have been measured, but it is not known how they generate force. We previously proposed that the DNA is not a passive substrate but that it plays an active role in force generation. This "scrunchworm hypothesis" holds that the motor proteins repeatedly dehydrate and rehydrate the DNA, which then undergoes cyclic shortening and lengthening motions. These are captured by a coupled protein-DNA grip-and-release cycle to rectify the motion and translocate the DNA into the capsid. In this study, we examined the interactions of dsDNA with the dodecameric connector protein of bacteriophage ϕ29, using molecular dynamics simulations on four different DNA sequences, starting from two different conformations (A-DNA and B-DNA). In all four simulations starting with the protein equilibrated with A-DNA in the channel, we observed transitions to a common, metastable, highly scrunched conformation, designated A*. This conformation is very similar to one recently reported by Kumar and Grubmüller in much longer MD simulations on B-DNA docked into the ϕ29 connector. These results are significant for four reasons. First, the scrunched conformations occur spontaneously, without requiring lever-like protein motions often believed to be necessary for DNA translocation. Second, the transition takes place within the connector, providing the location of the putative "dehydrator". Third, the protein has more contacts with one strand of the DNA than with the other; the former was identified in single-molecule laser tweezer experiments as the "load-bearing strand". Finally, the spontaneity of the DNA-protein interaction suggests that it may play a role in the initial docking of DNA in motors like that of T4 that can load and package any sequence. [ABSTRACT FROM AUTHOR]

We formulate a continuum approach to the equation of state (density dependence of osmotic pressure) of bulk DNA and encapsidated DNA, as well as review the phase diagram of DNA in the regime of densities relevant for DNA packing in bacteriophages. We derive the first integral of the equilibrium equations that connects the behavior of DNA in the bulk and in nanoscale enclosures, and we delineate the changes wrought upon the mesophase equilibria of encapsidated DNA. We show how multiphase equilibria and complicated spatial distribution of DNA density and orientation can emerge due to the curvature contribution to the DNA osmotic pressure within the capsid. [ABSTRACT FROM AUTHOR]

Protein search for specific sequences on DNA marks the beginning of major biological processes. Experiments indicate that proteins find and recognize their targets quickly and efficiently. Because of the large number of experimental and theoretical investigations, there is a reasonable understanding of the protein search processes in purified in vitro systems. However, the situation is much more complex in live cells where multiple biochemical and biophysical processes can interfere with the protein search dynamics. In this study, we develop a theoretical method that explores the effect of crowding on DNA chains during the protein search. More specifically, the role of static and dynamic obstacles is investigated. The method employs a discrete-state stochastic framework that accounts for most relevant physical and chemical processes in the system. Our approach also provides an analytical description for all dynamic properties. It is found that the presence of the obstacles can significantly modify the protein search dynamics. This effect depends on the size of the obstacles, on the spatial positions of the target and the obstacles, on the nature of the search regime, and on the dynamic nature of the obstacles. It is argued that the crowding on DNA can accelerate or slow down the protein search dynamics depending on these factors. A comparison with existing experimental and theoretical results is presented. Theoretical results are discussed using simple physical-chemical arguments, and they are also tested with extensive Monte Carlo computer simulations. [ABSTRACT FROM AUTHOR]

Ultrafast two-dimensional infrared (2D-IR) spectroscopy of a 15-mer A-T DNA duplex in solution has revealed structure-dependent vibrational coupling and energy transfer processes linking bases with the sugar-phosphate backbone. Duplex melting induces significant changes in the positions of off-diagonal peaks linking carbonyl and ring-stretching vibrational modes of the adenine and thymine bases with vibrations of the phosphate group and phosphodiester linkage. These indicate that Watson-Crick hydrogen bonding and helix formation lead to a unique vibrational coupling arrangement of base vibrational modes with those of the phosphate unit. On the basis of observations from time-resolved 2D-IR data, we conclude that rapid energy transfer processes occur between base and backbone, mediated by additional modes located on the deoxyribose moiety within the same nucleotide. These relaxation dynamics are insensitive to duplex melting, showing that efficient intramolecular energy relaxation to the solvent via the phosphate groups is the key to excess energy dissipation in both single- and double-stranded DNA. [ABSTRACT FROM AUTHOR]

We show that the dynamic nonequilibrium segmental concentration profile of a single nanochannel confined DNA molecule can be described via a partial differential evolution equation based on nonlinear diffusion, using an approach analogous to that used in the description of many-molecule systems such as polymer solutions. This equation can describe the segmental concentration profile of a single polymer along the nanochannel as a function of time for chain behavior ranging from states of high compression to equilibrium. In particular, to demonstrate the generality of our approach, we show that our model can describe two distinct types of experimental behavior generated via a sliding bead assay, symmetric relaxation resulting from free expansion of the polymer after compression, and the evolution of DNA concentration "shock waves" as a molecule is driven from equilibrium to a compressed state. [ABSTRACT FROM AUTHOR]

Modifications to nucleotides in the genome can lead to mutations or are involved in regulation of gene expression, and therefore, finding the site of modification is a worthy goal. Robust methods for sequencing modification sites on commercial sequencers have not been developed beyond the epigenetic marks on cytosine. Herein, a method to sequence DNA modification sites was developed that utilizes DNA glycosylases found in the base excision repair pathway to excise the modification. This approach yields a gap at the modification site that is sealed by T4-DNA ligase, yielding a product strand missing the modification. Upon sequencing, the modified nucleotide is reported as a deletion mutation, identifying its location. This approach was used to detect a uracil (U) or 8-oxo- 7,8-dihydroguanine (OG) in codon 12 of the KRAS gene in synthetic oligodeoxynucleotides. Additionally, an OG modification site was placed in the VEGF promoter in a plasmid and sequenced. This method requires only commercially available materials and can be put into practice on any sequencing platform, allowing this method to have broad potential for finding modifications in DNA. [ABSTRACT FROM AUTHOR]

The distorted and unique expression of microRNAs (miRNAs) in cancer makes them an attractive source of biomarkers. However, one of prerequisites for the application of miRNAs in clinical practice is to accurately profile their expression. Currently available assays normally require pre-enrichment, amplification, and labeling steps, and most of them are semiquantitative. In this study, we converted the signal of target miR-21 into reporter peptide by a DNA-peptide probe and the reporter peptide was ultimately quantified using LC-MS/MS-based targeted proteomics. Specifically, substrate peptide GDKAVLGVDPFR containing reporter peptide AVLGVDPFR and tryptic cleavage site (lysine at position 3) was first designed, followed by the conjugation with DNA sequence that was complementary to miR-21. The newly formed DNA-peptide probe was then hybridized with miR-21, which was biotinylated and attached to streptavidin agarose in advance. After trypsin digestion, the reporter peptide was released and monitored by a targeted proteomics assay. The obtained limit of quantification (LOQ) was 1 pM, and the detection dynamic range spanned ~5 orders of magnitude. Using this assay, the developed quasi-targeted proteomics approach was applied to determine miR-21 level in breast cells and tissue samples. Finally, qRT-PCR was also performed for a comparison. This report grafted the strategy of targeted proteomics into miRNA quantification. [ABSTRACT FROM AUTHOR]

This paper addresses a growing need in clinical diagnostics for parallel, multiplex analysis of biomarkers from small biological samples. It describes a new procedure for assembling arrays of ssDNA and proteins on paper. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper and proceeds to assemble microzones of DNAconjugated paper into arrays capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The synthesis of ssDNA oligonucleotides on paper is convenient and effective with 32% of the oligonucleotides cleaved and eluted from the paper substrate being full-length by HPLC for a 32-mer. These ssDNA arrays can be used to detect fluorophore-linked DNA oligonucleotides in solution, and as the basis for DNA-directed assembly of arrays of DNA-conjugated capture antibodies on paper, detect protein antigens by sandwich ELISAs. Paper-anchored ssDNA arrays with different sequences can be used to assemble paper-based devices capable of detecting DNA and antibodies in the same device and enable simple microfluidic paper-based devices. [ABSTRACT FROM AUTHOR]

Accurate detection of organisms is crucial for the effective management of threatened and invasive species because false detections directly affect the implementation of management actions. The use of environmental DNA (eDNA) as a species detection tool is in a rapid development stage; however, concerns about accurate detections using eDNA have been raised. We evaluated the effect of sampled water volume (0.25 to 2 L) on the detection rate for three macroinvertebrate species. Additionally, we tested (depending on the sampled water volume) what amount of total extracted DNA should be screened to reduce uncertainty in detections. We found that all three species were detected in all volumes of water. Surprisingly, however, only one species had a positive relationship between an increased sample volume and an increase in the detection rate. We conclude that the optimal sample volume might depend on the species-habitat combination and should be tested for the system where management actions are warranted. Nevertheless, we minimally recommend sampling water volumes of 1 L and screening at least 14 μL of extracted eDNA for each sample to reduce uncertainty in detections when studying macroinvertebrates in rivers and using our molecular workflow. [ABSTRACT FROM AUTHOR]

Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed. [ABSTRACT FROM AUTHOR]

Novel poly(l-lysine)-block-poly(l-proline) (PLL-b-PLP)-basedmaterials with all PLPhelical conformers, i.e., PLP II and the rare PLP I are here reported.Electrostatic supramolecular complexation of the adjacent cationicPLL with anionic molecules bearing DNA analogue H-bonding functionalities,such as deoxyguanosine monophosphate (dGMP), preserves the extendedPLP II helix, and the complexed molecule is locked and held in positionby orthogonal shape-persistent hydrogen-bonded dGMP ribbons and theirextended π-stacking. The branched anionic surfactant dodecylbenzenesulfonicacid (DBSA) on the other hand, introduces periodicity frustrationand interlayer plasticization, leading to a reversed mutarotationto the more compact PLP I helix by complexation, without externalstimuli, and is here reported for the first time. We foresee thatour findings can be used as a platform for novel molecularly adaptivefunctional materials, and could possibly give insight in many proline-relatedtransmembrane biological functions. [ABSTRACT FROM AUTHOR]

Novel multifunctional magnetic zirconium hexacyanoferrate nanoparticles (ZrHCF MNPs) were prepared, which consisted of magnetic beads (MBs) inner core and zirconium hexacyanoferrate(II) (ZrHCF) outer shell. As an artificial peroxidase, the ZrHCF MNPs exhibited remarkable electrocatalytic properties in the reduction of H2O2 at 0.2 V vs saturated calomel electrode (SCE). On the basis of the bonding interaction between Zr (IV) of the shell ZrHCF framework and phosphonate groups, the 5'-phosphorylated ssDNA probes with a consecutive stretch of guanines as a spacer could be incorporated in ZrHCF MNPs easily. Thus, DNA-grafted ZrHCF MNPs could be simply obtained by magnetic separation. The prepared nanoelectrocatalyst was further used as signal nanoprobe for the ultrasensitive electrochemical DNA assay. Under optimal conditions, the proposed biosensor presents high sensitivity for detecting target DNA with a linear range from 1.0 fM to 1.0 nM and a low detection limit of 0.43 fM. Moreover, it exhibits good performance with excellent selectivity, high stability, and acceptable fabrication reproducibility. [ABSTRACT FROM AUTHOR]

Free solution capillary electrophoresis (CE) is a useful technique for measuring the translational diffusion coefficients of charged analytes. The measurements are relatively fast if the polarity of the electric field is reversed to drive the analyte back and forth past the detection window during each run. We have tested the validity of the resulting diffusion coefficients using double-stranded DNA molecules ranging in size from 20 to 960 base pairs as the model system. The diffusion coefficients of small DNAs are equal to values in the literature measured by other techniques. However, the diffusion coefficients of DNA molecules larger than ∼30 base pairs are anomalously high and deviate increasingly from the literature values with increasing DNA molar mass. The anomalously high diffusion coefficients are due to electrostatic coupling between the DNA and its counterions. As a result, the measured diffusion coefficients vary with the diffusion coefficient of the counterion, as well as with cation concentration and electric field strength. These effects can be reduced or eliminated by measuring apparent diffusion coefficients of the DNA at several different electric field strengths and extrapolating the results to zero electric field. [ABSTRACT FROM AUTHOR]

High-yield preparation of ultrathin two-dimensional (2D) nanosheets is of great importance for the further exploration of their unique properties and promising applications. Herein, for the first time, the high-yield and scalable production of ultrathin 2D ternary chalcogenide nanosheets, including Ta2NiS5 and Ta2NiSe5, in solution is achieved by exfoliating their layered microflakes. The size of resulting Ta2NiS5 and Ta2NiS5 nanosheets ranges from tens of nanometers to few micrometers. Importantly, the production yield of single-layer Ta2NiS5 nanosheets is very high, ca. 86%. As a proof-of-concept application, the single-layer Ta2NiS5 is used as a novel fluorescence sensing platform for the detection of DNA with excellent selectivity and high sensitivity (with detection limit of 50 pM). These solution-processable, high-yield, large-amount ternary chalcogenide nanosheets may also have potential applications in electrocatalysis, supercapacitors, and electronic devices. [ABSTRACT FROM AUTHOR]

Thevibrational modes of the deoxyribose-phosphodiester backbonemoiety of DNA and their interactions with the interfacial aqueousenvironment are addressed with two-dimensional (2D) infrared spectroscopyon a femto- to picosecond time scale. Beyond the current understandingin the harmonic approximation, the anharmonic character and delocalizationof the backbone modes in the frequency range from 900 to 1300 cm–1are determined with both diagonal anharmonicitiesand intermode couplings on the order of 10 cm–1.Mediated by the intermode couplings, energy transfer between the backbonemodes takes place on a picosecond time scale, parallel to vibrationalrelaxation and energy dissipation into the environment. Probing structuraldynamics noninvasively via the time evolution of the 2D lineshapes,limited structural fluctuations are observed on a 300 fs time scaleof low-frequency motions of the helix, counterions, and water shell.Structural disorder of the DNA–water interface and DNA–waterhydrogen bonds are, however, preserved for times beyond 10 ps. Thedifferent interactions of limited strength ensure ultrafast vibrationalrelaxation and dissipation of excess energy in the backbone structure,processes that are important for the structural integrity of hydratedDNA. [ABSTRACT FROM AUTHOR]

The double-helical structures ofDNA are experimentally distinguishedby the circular dichroism (CD) spectra. The CD spectra are quite differentbetween the left- and right-handed double-helical structures of DNA.The lowest peak is negative for the left-handed Z-DNA but positivefor the right-handed B-DNA. Using the Z-DNA model with a strong stackinginteraction, we examined whether the CD spectra depend on the distancebetween the two base pairs, deoxy-guanosine (dG) and deoxy-cytidine(dC). The result showed that the feature of the SAC-CI CD spectrachanges from Z-DNA to B-DNA when increasing the distance between thetwo base pairs. Therefore, we concluded that the stacking interactionis the origin of the lowest negative peak, being the feature of theCD spectra of Z-DNA, and at the same time that the lack of the negativepeak at about 290–300 nm of the CD spectra of B-DNA is dueto the weak stacking interaction in B-DNA. [ABSTRACT FROM AUTHOR]

Helicases unwind double-stranded nucleic acids, remove secondary structures from single-stranded nucleic acids, and remove proteins bound to nucleic acids. For many helicases, the mechanisms for these different 3' functions share the ability to translocate with a directional bias as a result of ATP binding and hydrolysis. Nonstructural protein 3 (NS3) is an essential enzyme expressed by the hepatitis C virus (HCV) and is known to catalyze the unwinding of both DNA and RNA substrates in a 3'-to-5' direction. We investigated the role of nucleic acid binding in the unwinding mechanism by examining ATP-independent unwinding. We observed that even in the absence of ATP, the NS3 helicase domain (NS3h) unwound duplexes only when they contained a 3'-tail (i.e., 3'-to-5' directionality). Blunt-ended duplexes and 5'-tailed duplexes were not melted even in the presence of a large excess concentration of the protein. NS3h was found to diffuse rapidly along single-stranded DNA at a rate of 30 nucleotides2 s-1. Upon encountering an appropriate single-strand/double-strand (ss/ds) junction, NS3h slowly melted the duplex under conditions with an excess protein concentration relative to DNA concentration. When a biotin-streptavidin block was placed into the ssDNA region, no melting of DNA was observed, suggesting that NS3h must diffuse along the ssDNA, and that the streptavidin blocked the diffusion. We conclude that the specific interaction between NS3h and the ss/dsDNA junction, coupled with diffusion, allows binding energy to melt duplex DNA with a directional bias. Alternatively, we found that the frill-length NS3 protein did not exhibit strict directionality and was dependent on duplex DNA length. NS3 was able to unwind the duplex even in the presence of the biotin-streptavidin block. We propose a noncanonical model of unwinding for NS3 in which the enzyme binds directly to the duplex via protein-protein interactions to melt the substrate. [ABSTRACT FROM AUTHOR]

Interstrand cross-links in cellular DNA are highly deleterious lesions that block transcription and replication. We recentiy characterized two new structural types of interstrand cross-links derived from the reaction of abasic (Ap) sites with either guanine or adenine residues in duplex DNA Interestingly, these Ap-derived cross-links are forged by chemically reversible processes, in which the two strands of the duplex are joined by hemiaminal, imine, or aminoglycoside linkages. Therefore, understanding the stability of Ap-derived cross-links may be critical in defining the potential biological consequences of these lesions. Here we employed bacteriophage cp29 DNA polymerase, which can couple DNA synthesis and strand displacement, as a model system to examine whether dA-Ap cross-links can withstand DNA-processing enzymes. We first demonstrated that a chemically stable interstrand cross-link generated by hydride reduction of the dG-Ap cross-link completely blocked primer extension by ϕ29 DNA polymerase at the last unmodified nucleobase preceding cross-link. We then showed that the nominally reversible dA-Ap cross-link behaved, for all practical purposes, like an irreversible, covalent DNA-DNA cross-link. The dA-Ap cross-link completely blocked progress of the ϕ29 DNA polymerase at the last unmodified base before the cross-link. This suggests that Ap-derived cross-links have the power to block various DNA-processing enzymes in the cell. In addition, our results reveal (ϕ29 DNA polymerase as a tool for detecting the presence and mapping the location of interstrand cross-links (and possibly other lesions) embedded within regions of duplex DNA [ABSTRACT FROM AUTHOR]

Tyrosyl-DNA phosphodiesterase I (Tdp1) is a DNA repair enzyme conserved across eukaryotes that catalyzes the hydrolysis of the phosphodiester bond between the tyrosine residue of topoisomerase I and the 3'-phosphate of DNA. Atomic level details of the mechanism of Tdpl are proposed and analyzed using a fully quantum mechanical, geometrically constrained model. The structural basis for the computational model is the vanadate-inhibited crystal structure of human Tdp1 (hTdp1, Protein Data Bank entry 1RFF). Density functional theory computations are used to acquire thermodynamic and kinetic data along the catalytic pathway, including the phosphoryl transfer and subsequent hydrolysis. Located transition states and intermediates along the reaction coordinate suggest an associative phosphoryl transfer mechanism with five-coordinate phosphorane intermediates. Similar to both theoretical and experimental results for phospholipase D, the proposed mechanism for hTdpl also includes the thermodynamically favorable possibility of a four-coordinate phosphohistidine "dead-end" product. [ABSTRACT FROM AUTHOR]

Glassy carbon electrodes were modified with small carbon nano-onions (CNOs) and activated by electrografting of diazonium salts bearing terminal carboxylic acid and maleimide groups. The CNO-modified surfaces were characterized by ESEM and AFM microscopy as well as by electrochemical techniques. The modified electrodes were used for the amperometric detection of a model DNA target sequence associated with the human papillomavirus by immobilizing short recognition sequences by amidation or thiol-maleimide reactions. The analytical parameters of the developed biosensors were compared with glassy carbon electrodes without CNOs. In both cases, the incorporation of CNOs resulted in an enhancement in sensitivity and a decrease in detection limits ascribed to a combination of large surface areas and enhanced electron transfer properties of the CNO-modified electrodes. These results offer promise for the construction of other CNO-based biomolecule detection platforms with enhanced sensitivities. [ABSTRACT FROM AUTHOR]

DNAdamage is ubiquitous in all mammalian cells with the occurrenceof more than 60 000 times per day per cell. In particular,DNA damage in neurons is found to accumulate with age and has beensuggested to interfere with the synthesis of functional proteins.Moreover, recent studies have found through transgenic mice that humanamyloid precursor protein causes an increase in DNA double-strandbreaks (DSBs) with the effect of a prolongation in DNA repair. Itis surmised that amyloid β (Aβ) exacerbates the DNA DSBsin neurons, possibly engendering neuronal dysfunction as a result.However, a good understanding on the holistic interaction mechanismsand the manner in which Aβ intertwines with DNA damage is stillin its infancy. In our study, we found that DNA with an AT-rich sequencehas a very low binding affinity toward Aβ by means of moleculardynamics simulation. While we have pursued a particular sequence ofDNA in this study, other DNA sequences are expected to affect theinteraction and binding affinity between DNA and Aβ, and willbe pursued in our further research. Nonetheless, we have uncoveredfavorable interaction between the positively charged side chain ofAβ and the two ends of DNA. The latest experiment reveals thatmany of the double-stranded breaks in neurons can be fixed via DNArepair mechanisms but not in the case that Aβs are present.It is found that the increased numbers of DSBs prevail in active neurons.Here, on the basis of the favorable interaction between Aβ andthe two ends of DNA, we propose the possibility that Aβ preventsDNA repair via binding directly to the break ends of the DNA, whichfurther exacerbates DNA damage. Moreover, we have found that the basepair oxygen of the DNA has a greater preference to form hydrogen bondsthan the backbone oxygen with Aβ at the two ends. Thus, we postulatethat Aβ could serve to prevent the repair of AT-rich DNA, andit is unlikely to cause its breakage or affect its binding towardhistone. Another important observation from our study is that AT-richDNA has very little or no influence on Aβ oligomerization. Finally,even though we do not observe any dramatic DNA conformational changein the presence of Aβ, we do observe an increase in diversityof the DNA structural parameters such as groove width, local basestep, and torsional angles in lieu of Aβ interactions. [ABSTRACT FROM AUTHOR]

Hypoxiaa hallmark of solidtumorsmakes hypoxiccells radioresistant. On the other hand, DNA, the main target of anticancertherapy, is not sensitive to the near UV photons and hydrated electrons,one of the major products of water radiolysis under hypoxic conditions.A possible way to overcome these obstacles to the efficient radio-and photodynamic therapy of cancer is to sensitize the cellular DNAto electrons and/or ultraviolet radiation. While incorporated intogenomic DNA, modified nucleosides, 5-bromo-2′-deoxyuridinein particular, sensitize cells to both near-ultraviolet photons andγ rays. It is believed that, in both sensitization modes, thereactive nucleobase radical is formed as a primary product which swiftlystabilizes, leading to serious DNA damage, like strand breaks or cross-links.However, despite the apparent similarity, such radio- and photosensitizationof DNA seems to be ruled by fundamentally different mechanisms. Inthis review, we demonstrate that the most important factors decidingon radiodamage to the labeled DNA are (i) the electron affinity (EA)of modified nucleoside (mNZ), (ii) the local surroundings of the labelthat significantly influences the EA of mNZ, and (iii) the strengthof the chemical bond holding together the substituent and a nucleobase.On the other hand, we show that the UV damage to sensitized DNA isgoverned by long-range photoinduced electron transfer, the efficiencyof which is controlled by local DNA sequences. A critical review ofthe literature mechanisms concerning both types of damage to the labeledbiopolymer is presented. Ultimately, the perspectives of studies onDNA sensitization in the context of cancer therapy are discussed. [ABSTRACT FROM AUTHOR]

N6-(2-Deoxy-D-erytfiro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG) arises from N7-methylation of deoxyguanosine followed by imidazole ring opening. The lesion has been reported to persist in animal tissues. Previous in vitro replication bypass investigations of the MeFapy-dG adduct revealed predominant insertion of C opposite the lesion, dependent on the identity of the DNA polymerase (Pol) and the local sequence context. Here we report crystal structures of ternary Pol-DNA-dNTP complexes between MeFapy-dG-adducted DNA template: primer duplexes and the Y-family polymerases human Pol η and P2 Pol IV (Dpo4) from Sulfolobus solfataricus. The structures of the hPol η and Dpo4 complexes at the insertion and extension stages, respectively, are representative of error-free replication, with MeFapy-dG in the anti conformation and forming Watson--Crick pairs with dCTP or dC. [ABSTRACT FROM AUTHOR]

We have investigated the action of the human DNA polymerase ε (hpol ε ) and η (hpol η) catalytic cores on Gquadruplex (G4) DNA substrates derived from the promoter of the c-MYC proto-oncogene. The translesion enzyme hpol η exhibits a 6.2-fold preference for binding to G4 DNA over non- G4 DNA, while hpol ε binds both G4 and non-G4 substrates with nearly equal affinity. Kinetic analysis of single-nucleotide insertion by hpol η reveals that it is able to maintain >25% activity on G4 substrates compared to non-G4 DNA substrates, even when the primer template junction is positioned directly adjacent to G22 (the first tetrad-associated guanine in the c-MYC G4 motif). Surprisingly, hpol η fidelity increases ~ 15-fold when copying G22. By way of comparison, hpol ε retains ~4% activity and has a 33-fold decrease in fidelity when copying G22. The fidelity of hpol η is ~ 100-fold greater than that of hpol ε when comparing the misinsertion frequencies of the two enzymes opposite a tetrad-associated guanine. The kinetic differences observed for the B- and Y-family pols on G4 DNA support a model in which a simple kinetic switch between replicative and TLS pols could help govern fork progress during G4 DNA replication. [ABSTRACT FROM AUTHOR]

A novel "signal-on" photoelectrochemical (PEC) biosensor for sensitive detection of human T-cell lymphotropic virus type II (HTLV-Il) DNA was developed on the basis of enzymatic amplification coupled with terminal deoxynucleotidyl transferase (TdT)-mediated extension strategy. The intensity of the photocurrent signal was proportional to the concentration of the HTLV-II DNA-target DNA (tDNA) by dual signal amplification. In this protocol, GRCdS: Mn/ZnS nanocomposites were used as photoelectric conversion material, while pDNA was used as the tDNA recognizing unit Moreover, the TdT-mediated extension and the enzymatic signal amplification technique were used to enhance the sensitivity of detection. Using this novel dual signal amplification strategy, the prototype of PEC DNA sensor can detect as low as ∼0.033 fM of HTLV-II DNA with a linear range of 0.1-5000 fM, with excellent differentiation ability even for single-base mismatches. This PEC DNA assay opens a promising platform to detect various DNA targets at ultralow levels for early diagnoses of different diseases. [ABSTRACT FROM AUTHOR]

Metastasis, the capability of tumor cells to spread and grow at distant sites, is the primary factor in cancer mortality. Because metastasis in sentinel lymph nodes suggests the original spread of tumors from a primary site, the detection of lymph node involvement with cancer serves as an important prognostic and treatment parameter. Here we have developed a panel of DNA aptamers specifically binding to colon cancer cell SW620 derived from metastatic site lymph node, with high affinity after 14 rounds of selection by the cell-SELEX (systematic evolution of ligands by exponential enrichment) method. The binding affinities of selected aptamers were evaluated by flow cytometry. Aptamer XL-33 with the best binding affinity (0.7 nM) and its truncated sequence XL-33-1 with 45 nt showed excellent selectivity for recognizing target cell SW620. The binding entity of the selected aptamer has been preliminarily determined as a membrane protein on the cell surface. Tissue imaging results showed that XL-33-1 was highly specific to the metastatic tumor tissue or lymph node tissue with corresponding cancer metastasis and displayed an 81.7% detection rate against colon cancer tissue with metastasis in regional lymph nodes. These results suggest that XL-33-1 has great potential to become a molecular imaging agent for early detection of lymph node tissue with colon cancer metastasis. More importantly, this study clearly demonstrates that DNA ligands selectively recognizing metastatic cancer cells can be readily generated by metastatic-cell-based SELEX for potential applications in metastatic cancer diagnosis and treatment. [ABSTRACT FROM AUTHOR]

We report a novel strategy for regiospecific hetero-assembly of DNA-modified gold nanoparticles (DNA-AuNPs) onto upconversion nanoparticles (UCNPs) into hybrid lab-on-a-particle systems. The DNA-AuNPs have been assembled onto the hexagonal plate-like UCNPs with well-regulated stoichiometry and controlled organization onto the different facets of UCNP, forming various addressable superstructures. The fine-tuning of stoichiometry and organization is realized by biorecognition specificity of DNA toward specific crystal facets of UCNPs. Such a hetero-assembled DNA-AuNP/UCNP system maintains both plasmonic resonance of AuNPs and fluorescent properties of UCNPs, allowing targeted dual-modality imaging of cancer cells using an aptamer. [ABSTRACT FROM AUTHOR]

Nature owns remarkable capabilities in sensing target molecules, while the artificial biosensor lags far behind nature. Inspired by nature, we devise a new sensing platform that can specifically bind the molecules and synchronously initiate a specific signal response. We rationally designed a type of bipolar probe that is comprised of a hydrophilic DNA part and a hydrophobic conjugated polymer (CP) unit. In aqueous solution, they can form micelles with a hydrophobic CP core and a hydrophilic DNA shell. The aggregation-caused quenching suppresses the fluorescence of CP. Adding telomerase, the hydropathical profile of the bipolar probes is drastically regulated that results in the collapse of micelles and liberates fluorescence simultaneously. The probe has been used in both mimic systems and real urine samples (38 samples). We achieve sensitive and specific detection of telomerase and obtain clearly classification for normal people and cancer patients. It can also be used in a signal off sensor that is used to detect mercury ions. [ABSTRACT FROM AUTHOR]

The genomes of all cells are intimately associated with proteins, which are important for compaction, scaffolding, and gene regulation. Here we show that pre-existing protein-DNA complexes (roadblocks) diminish and--interestingly--enhance the ability of particular sequence-specific proteins to move along DNA to locate their binding sites. We challenge the bacterial DNA adenine methyltransferase (Dam, recognizes 5'-GATC-3') with tightly bound EcoRV ENase-DNA complexes, which bend DNA. A single EcoRV roadblock does not alter processive (multiple modifications) methylation by Dam. This result disfavors a reliance on heavily touted mechanisms involving sliding or short hops for Dam. Specific conformations of two EcoRV roadblocks cause an increase in processivity. The histone-like leucine-responsive regulatory protein (Lrp) binds DNA nonspecifically as an octamer, and also increases Dam's processivity. These results can be explained by our prior demonstration that Dam moves over large regions (>300 bp) within a single DNA molecule using an "intersegmental hopping" mechanism. This mechanism involves the protein hopping between looped DNA segments. Both roadblock systems can cause the DNA to loop and therefore facilitate intersegmental hopping. For Lrp, this only occurs when the Dam sites are separated (by >134bp) such that they can be looped around the protein. Intersegmental hopping may well be a general mechanism for proteins that navigate long distances along compacted DNA Unlike Dam, EcoRl ENase (recognizes 5'-GAATTC-3') relies extensively on a sliding mechanism, and as expected, Lrp decreases its processivity. Our systematic use of protein roadblocks provides a powerful strategy to differentiate between site location mechanisms. [ABSTRACT FROM AUTHOR]

Hydrolysis of the N-glycosyl bond between a nucleobase and deoxyribose leaves an abasic site within duplex DNA. The abasic site can react with exocyclic amines of nucleobases on the complementary strand to form interstrand DNA-DNA cross-links (ICLs). We find that several enzymes from the base excision repair (BER) pathway protect an abasic site on one strand of a DNA duplex from cross-linking with an amine on the opposing strand. Human alkyladenine DNA glycosylase (AAG) and Escherichia coli 3-methyladenine DNA glycosylase II (AlkA) accomplish this by binding tightly to the abasic site and sequestering it. AAG protects an abasic site opposite T, the product of its canonical glycosylase reaction, by a factor of ~10-fold, as estimated from its inhibition of the reaction of an exogenous amine with the damaged DNA. Human apurinic/apyrimidinic site endonuclease 1 and E. coli endonuclease III both decrease the amount of ICL at equilibrium by generating a single-strand DNA nick at the abasic position as it is liberated from the cross-link. The reversibility of the reaction between amines and abasic sites allows BER enzymes to counter the potentially disruptive effects of this type of cross-link on DNA transactions. [ABSTRACT FROM AUTHOR]

A DNA G-quadruplex (G4) formed at the oncogene c-MYC promoter region functions as a gene silencer. Due to its high stability at physiological K+ concentrations, its thermodynamics and kinetic properties have not been characterized in physiological solution conditions. In this work, we investigated the unfolding and folding transitions of single c-MYC G4 and several of its truncated or point mutants at 100 mM KCl concentration under mechanical force. We found that the wild type could fold into multiple species, and the major specie has a slow unfolding rate of (1.4 ± 1.0) x 10-6 s-1. The force-dependent thermodynamics and kinetic properties of the major specie were obtained by studying a truncated mutant, Myc2345, that contains the G-tracts 2, 3, 4, and 5. As the c-MYC G4 is a prototype of many other intermolecular parallel-stranded G4's, our results provide important insights into the stability of a broad class of promoter G4's which also play a role in transcription regulation and are potential anticancer targets. [ABSTRACT FROM AUTHOR]

Chirality is one of the fundamental biochemical properties in a living system, and a lot of biological and physiological processes are greatly influenced by the chirality of molecules. Inspired by this phenomenon, we study the covalent assembly of DNA on chiral molecule modified surfaces and further discuss the hybridization of DNA on chiral surfaces with nucleic acids. Take methylene blue (MB) modified DNA as a model molecule, we show that the peak current of the LNIBC (NIBC, N-isobutyryl-L(D)-cysteine) modified gold surface (L-surface) is larger than the D-surfece because of a stronger interaction between short-chain DNA and the Lsurface; however, the D-surface has a higher hybridization efficiency than the L-surface. Moreover, we apply this result to actual application by choosing an electrochemical DNA (EDNA) sensor as a potential platform. Furthermore, we further amplify the difference of hybridization efficiency using the supersandwich assay. More importantly, our findings are successfully employed to program the sensitivity and limit of detection. [ABSTRACT FROM AUTHOR]

Hydrogen atom abstraction from the C5'-position of nucleotides in DNA results in direct strand scission by generating alkali-labile fragments from the oxidized nucleotide. The major damage consists in a terminus containing a 5'-aldehyde as part of an otherwise undamaged nucleotide. Moreover it is considered as a polymorphic DNA strand break lesion since it can be borne by any of the four nucleosides encountered in DNA. Here we propose an expeditious synthesis of oligonucleotides (ON) ending with this 5'-aldehyde group (5'-AODN). This straightforward and cheap strategy relies on Pfitzner--Moffatt oxidation performed on solid support followed by a transient protection of the resulting aldehyde function. This method is irrespective of the 5'-terminal nucleobase and most interestingly can be directly extended to RNA to produce the corresponding 5'-AORN. We also report preliminary results on recognition of 5'-AODN by base excision repair (BER) enzymes. [ABSTRACT FROM AUTHOR]

The ability to detect DNA modification sites at single base resolution could significantly advance studies regarding DNA adduct levels, which are extremely difficult to determine. Artificial nucleotides that are specifically incorporated opposite a modified DNA site offer a potential strategy for detection of such sites by DNA polymerase-based systems. Here we investigate the action of newly synthesized base-modified benzimidazole-derived 2'-deoxynucleoside-5'-O-triphosphates on DNA polymerases when performing translesion DNA synthesis past the pro-mutagenic DNA adduct O6-benzylguanine (O6-BnG). We found that a mutated form of KlenTaq DNA polymerase, i.e., KTqM747K, catalyzed O6-BnG adduct-specific processing of the artificial BenziTP in favor of the natural dNTPs. Steady-state kinetic parameters revealed that KTqM747K catalysis of BenziTP is 25-fold more efficient for template O6-BnG than G, and 5-fold more efficient than natural dTMP misincorporation in adduct bypass. Furthermore, the nucleotide analogue BenziTP is required for full-length product formation in O6-BnG bypass, as without BenziTP the polymerase stalls at the adduct site. By combining the KTqM747K polymerase and BenziTP, a first round of DNA synthesis enabled subsequent amplification of Benzi-containing DNA. These results advance the development of technologies for detecting DNA adducts. [ABSTRACT FROM AUTHOR]

A growing body of evidence suggests that hydrogen peroxide (H2O2) plays an active role in the regulation of various physiological processes. Development of sensitive probes for H2O2 is an urgent work. In this study, we proposed a DNA-mediated silver nanoparticle and graphene quantum dot hybrid nanocomposite (AgNPDNA< 2>GQps) for sensitive fluorescent detection of H2O2. The sensing mechanism is based on the etching effect of H2O2 to AgNPs and the cleavage of DNA by as-generated hydroxyl radicals (OH). The formation of AgNP-DNA(2)GQPs nanocomposite can result in fluorescence quenching of GQDs by AgNPs through the resonance energy transfer. Upon H2O2 addition, the energy transfer between AgNPs and GQDs mediated by DNA was weakened and obvious fluorescence recovery of GQDs could be observed. It is worth noting that the reaction product OH between H2O2 and AgNPs could cleave the DNAbridge and result in the disassembly of AgNP-DNA{®GQDs to achieve further signal enhancement With optimal conditions, the approach achieves a low detection limit of 0.10 μM for H2O2. Moreover, this nanocomposite is further extended to the glucose sensing in human urine combining with glucose oxidase (GOx) for the oxidation of glucose and formation of H2O2. The glucose concentrations in human urine are detected with satisfactory recoveries of 94.6-98.8% which holds potential for ultrasensitive quantitative analysis of glucose and supplies valuable information for diabetes mellitus research and clinical diagnosis. [ABSTRACT FROM AUTHOR]

Biosensing platforms are ideal for addressing the diagnostic needs of resource-poor areas; however, the translation of such systems from the laboratory to the point-of-need has been a slow process. Rapid prototyping methods that enable an application-specific biosensor to be created in a matter of hours from design tofabrication would expedite the clinical and field testing of such systems. Here, we demonstrate a benchtop method based on craft cutting and polymer-induced wrinkling for creating multiplexed electrochemical DNA biosensors. This fabrication method allows multiscale wrinkled electrodes with features in the millimeter to nanometer length scales to be created in a matter of hours. These wrinkled electrodes display an enhanced surface area compared to planar electrodes and are shown to be structurally tunable by changing the film thickness. We demonstrate that structural tunability of these electrodes is translatable tofunctional tunability as the density of surface-immobilized probe molecules can be manipulated using wrinkled electrodes of different thicknesses. Furthermore, a simple proof-of-concept electrocatalytic DNA biosensor is demonstrated for distinguishing between complementary and noncomplementary oligonucleotides. [ABSTRACT FROM AUTHOR]

Steadily growing demands for identification and 1 .Chip quantification of cellular metabolites in higher throughput have brought a need for new analytical technologies. Here, we developed a synthetic biological sensor system for quantifying metabolites from biological cell samples. For this, bacterial transcription factors were exploited, which bind to or dissociate from regulatory DNA elements in response to physiological changes in the cellular metabolite concentration range. Representatively, the bacterial pyruvate dehydrogenase (PdhR), trehalose (TreR), and L-arginine (ArgR) repressor proteins were functionalized to detect pyruvate, trehalose-6-phosphate (T6P), and arginine concentration in solution. For each transcription factor the mutual binding behavior between metabolite and DNA, their working range, and othogonality were determined. High-throughput, parallel processing, and automation were achieved through integration of the metabolic sensor system on a microfluidic large-scale integration (mLSI) chip platform. To demonstrate the functionality of the integrated metabolic sensor system, we measured diurnal concentration changes of pyruvate and the plant signaling molecule T6P within cell etxracts of Arabidopsis thaliana rosettes. The transcription factor sensor system is of generic nature and extendable on the microfluidic chip. [ABSTRACT FROM AUTHOR]

A layered MoS2-thionin composite was prepared by sonicating their mixture in an ionic liquid and gradient centrifugation. Because DNA is rarely present in single-stranded form, either naturally or after PCR amplification, the composite was used for fabrication of a double-stranded DNA (dsDNA) electrochemical biosensor due to stable electrochemical response, intercalation, and electrostatic interaction of thionin with DNA. The linear range over dsDNA concentration from 0.09 ng mL-1 to 1.9 ng mL-1 is obtained, and moreover, it is suitable for the detection of single-stranded DNA (ssDNA). The biosensor has been applied to the detection of circulating DNA from healthy human serum, and satisfactory results have been obtained. The constructed DNA electrochemical biosensor shows potential application in the fields of bioanalysis and clinic diagnosis. Furthermore, this work proposes a new method to construct electrochemical biosensors based on MoS2 sheets. [ABSTRACT FROM AUTHOR]

Contamination of heavy metal ions in an aquatic environment poses a serious threat to human health. More seriously, heavy metal ions are usually present in the environment in a mixture, and the synergetic toxicity of multiple heavy metal ions is revealed (Aragay et al. Chem. Rev. 2011, 111, 3433; Chu et al. Aquat Toxicol. 2002, 61, 53). Unfortunately, most of the existing methods based on DNA sequences are focusing on the detection of one type of metal ions. Simple and multiplexed detection of multiple metal ions has been poorly investigated and remains challenging. Here, we re-engineered the DNA sequences for Pb2+, Hg2+, and Ag+, through which the binding of multiple metal ions initiated the selfassembly of these DNA sequences. On the basis of our rationally designed multicolor fluorescent labeling of the DNA sequences, cascade fluorescence resonance energy transfer (FRET) occurred. As a result, a fingerprint fluorescent spectrum was produced to indicate the presence of a single type of metal ions or multiple metal ions. The major advantages of our cascade FRET fingerprint technology include the following: (1) the "mix and read" detection mode in homogeneous solution is simple without the need of complicated instruments; (2) only single excitation is required to provide the cascade FRET fingerprint spectrum; (3) multiplexed detection capability can be realized intuitively and sensitively. [ABSTRACT FROM AUTHOR]

DNA sequences that can form G-quadruplexes (G4s) are highly prevalent in the genome. However, the structures and functions of most G4-forming sequences in the genome are poorly understood. Therefore, the development of molecular probes for G4 recognition in biological samples, especially probes with long wavelength, are important for the basic research of G4s. Squaraines dyes exhibit sharp and intense absorption and strong emission in the red to NIR region, but very few of them have been reported as probes for the recognition of nucleic acids. Here we report the interactions of two squaraine dyes, STS and CSTS, with different kinds of DNA The dicyanomethylene-functionalized squaraine dye, CSTS, exhibits strong interaction with the parallel G4s, but no interaction with other DNA In aqueous conditions, this interaction causes the transformation of CSTS from H-aggregates to monomers, which results in decline and growth of the absorption spectra of both forms. The parallel G4s enhance the fluorescence of both STS and CSTS, but the fluorescence enhancement of CSTS is much higher than that of STS. CSTS is demonstrated to bind to G4s through end-stacking model on G-quartet surface. The high selectivity of CSTS to parallel G4s is attributed to its V-shaped rigid planar n scaffold. The high selectivity, very low background fluorescence, large absorption coefficient, and high fluorescence quantum yield make CSTS hold great promise as a long-wavelength probe for parallel G4 detection in biological samples or in vivo. [ABSTRACT FROM AUTHOR]