Reactivity between acetone and single-stranded DNA containing a 5′-capped 2′-fluoro-N7-methyl guanine

DNA-mediated catalysis is an emerging field in bioorganic chemistry and chemical biology. However, the functional group diversity and known reactivity of DNA (A, T, C, and G) is relatively limited in scope. This relatively defined reactivity can limit the utility of DNA as a catalyst. In an effort to expand the functional group diversity and chemical reactivity of DNA, we sought to explore reactions involving single-stranded DNA equipped with a stabilized variant of N7-methyl guanine (2′-fluoro-5′-N7-methyl guanine). Here, we show that 5′-capped 2′-fluoro-N7-methyl guanine-labeled single-stranded DNA reacts with a ketone to afford a ketone-labeled DNA. This reaction likely proceeds through a reactive ylide or N-heterocyclic carbene. Taken together, our findings suggest that 2′-fluoro-5′-N7-methyl guanine is a stable adduct that can be selectively incorporated into ssDNA and functionalized with a ketone moiety by reaction with a simple ketone. Incorporation of this nucleoside into ssDNA may be useful for the evolution of novel deoxyribozymes that catalyze new reactions, including those which proceed via a reactive ylide or N-heterocyclic carbene-mediated chemistry.     

Renewable microcolumns for automated DNA purification and flow-through amplification: from sediment samples through polymerase chain reaction

There is an increasing need for field-portable systems for the detection and characterization of microorganisms in the environment. Nucleic acids analysis is frequently the method of choice for discriminating between bacteria in complex systems, but standard protocols are difficult to automate and current microfluidic devices are not configured specifically for environmental sample analysis. In this report, we describe the development of an integrated DNA purification and polymerase chain reaction (PCR) amplification system and demonstrate its use for the automated purification and amplification of Geobacter chapellei DNA (genomic DNA or plasmid targets) from sediments. The system includes renewable separation columns for the automated capture and release of microparticle purification matrices, and can be easily reprogrammed for new separation chemistries and sample types. The DNA extraction efficiency for the automated system ranged from 3 to 25%, depending on the length and concentration of the DNA target. The system was more efficient than batch capture methods for the recovery of dilute genomic DNA even though the reagent volumes were smaller than required for the batch procedure. The automated DNA concentration and purification module was coupled to a flow-through, Peltier-controlled DNA amplification chamber, and used to successfully purify and amplify genomic and plasmid DNA from sediment extracts. Cleaning protocols were also developed to allow reuse of the integrated sample preparation system, including the flow-through PCR tube.     

FINDER: A Fluidly Confined CRISPR-Based DNA Reporter on Living Cell Membranes for Rapid and Sensitive Cancer Cell Identification

The accurate, rapid, and sensitive identification of cancer cells in complex physiological environments is significant in biological studies, personalized medicine, and biomedical engineering. Inspired by the naturally confined enzymes on fluid cell membranes, a f luidly confin ed CRISPR-based D NA reporter (FINDER) was developed on living cell membranes, which was successfully applied for rapid and sensitive cancer cell identification in clinical blood samples. Benefiting from the spatial confinement effect for improved local concentration, and membrane fluidity for higher collision efficiency, the activity of CRISPR-Cas12a was, for the first time, found to be significantly enhanced on living cell membranes. This new phenomenon was then combined with multiple aptamer-based DNA logic gate for cell recognition, thus a FINDER system capable of accurate, rapid and sensitive cancer cell identification was constructed. The FINDER rapidly identified target cells in only 20 min, and achieved over 80 % recognition efficiency with only 0.1 % of target cells presented in clinical blood samples, indicating its potential application in biological studies, personalized medicine, and biomedical engineering.     

Synthesis,characterization and interaction with DNA of mononuclear metal complexes with oxolinic acid

Seven new neutral mononuclear metal complexes of VO²⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Zn²⁺ and Cd²⁺ with the quinolone antibacterial agent oxolinic acid (=Hoxo) have been prepared and characterized with physicochemical and spectroscopic techniques. In all the complexes, oxolinic acid acts as a bidentate deprotonated ligand bound to the metal through the pyridone oxygen and one carboxylate oxygen. The metals in all the complexes are six-coordinate with slightly distorted octahedral geometry. The lowest energy model structures of the complexes Fe(oxo)₃, VO(oxo)₂(H₂O) and Mn(oxo)₂(H₂O)₂ have been determined with molecular modeling calculations. The ability of all the complexes to bind to calf-thymus DNA has been investigated with diverse spectroscopic techniques.     

Direct immobilization in poly(dimethylsiloxane) for DNA, protein and enzyme fluidic biochips

A new arraying method is presented based on the properties of poly(dimethylsiloxane) (PDMS) polymer to entrap beads bearing biologically active compounds. It is shown that such beads could be spotted and dried at the surface of a poly(vinyl chloride) master and subsequently transferred at the PDMS interface by direct moulding of the polymer on the mask. Moreover, the use of the PDMS-assisted-immobilization enables the development of either a low density array (100 spots) or a micro-channel biochip with a direct incorporation of the sensing element in a fluidic system for the quantitative detection of enzyme substrates, antigens and oligonucleotides, depending on the immobilized sensing element. All biochip formats were revealed by a chemiluminescent reaction detected with a charge coupled device camera.As a result, arrays of beads bearing active enzymes, antibodies and oligonucleotides were successfully obtained and enabled the achievement of biochips for the chemiluminescent detection of enzyme substrates, protein antigens and oligonucleotides sequence with detection limit of 1 μM, 1.5×10⁷ molecules and 10⁸ molecules, respectively.     

A Polymer‐Based Electrochemical DNA Biosensor for Salmonella: Preparation,Characterization and Calibration

In this paper, we present the results of the use of bifunctional polymeric films of polystyrene (10.3 KD–49.5 KD) to anchor oligo sequences of various lengths (15, 35 and 70‐mer). The polymers were prepared by radical polymerization with 4,4′‐azobis(4‐cyanovaleric acid) as initiator and 3‐Carboxy PROXYL to control the molecular weight and polydispersity. They were further modified with N‐hydroxysuccinimide to anchor the (5′‐AmMC₁₂) oligos. The anchoring reaction was done on a polymer‐modified glassy carbon electrode. The probes were hybridized with their ferrocene‐labeled complementary sequences. The hybridization reaction was followed by Osteryoung square wave voltammetry (OSWV). The calibration curve showed a narrow and sharp linear range between (5.7–8.0)×10^?7 M and a detection limit around 0.55 µM.     

The Charge Transfer Complex between 2, 3-diamino-5-bromopyridine and Chloranilic acid: Preparation,Spectroscopic Characterization,DNA binding,and DFT/PCM analysis

A charge transfer hydrogen bonded complex was prepared and experimentally explored in an acetonitrile (ACN) medium between the proton acceptor (electron donor) 2, 3-Diamino-5-bromopyridine and the proton donor (electron acceptor) chloranilic acid. The stoichiometry of the charge transfer complex is 1:1. The Benesi-Hildebrand equation is used to calculate the molar absorptivity (ε_CT), association constant (K_CT) and other spectroscopic physical characteristics. The solid compound was synthesized and studied using several spectroscopic methods. The presence of charge and proton transfers in the resultant complex was supported by ¹H NMR, FT-IR and SEM-EDX investigations. The complex DNA binding ability was investigated using electron absorption spectroscopy, and the CT complex binding mechanism is intercalative. The intrinsic binding constant (K_b) value is 5.2 × 10⁶M?1. The good binding affinity of the CT complex makes it potentially suitable for usage as a pharmaceutical in the future. Molecular docking calculations have been performed between CT complex and DNA (ID = 1BNA) to study the CT-DNA interaction theoretically. To corroborate the experimental findings, calculations based on DFT were carried out in the gas and PCM analysis where the existence of charge and hydrogen transfers. Finally, good agreement between experimental and theoretical computations was observed confirming that the basis set used is appropriate for the system under examination.     

A comparison between petrous bone and tooth,femur and tibia DNA analysis from degraded skeletal remains

Skeletal remains are the only biological material that remains after long periods; however, environmental conditions such as temperature, humidity, and pH affect DNA preservation, turning skeletal remains into a challenging sample for DNA laboratories. Sample selection is a key factor, and femur and tooth have been traditionally recommended as the best substrate of genetic material. Recently, petrous bone (cochlear area) has been suggested as a better option due to its DNA yield. This research aims to evaluate the efficiency of petrous bone compared to other cranium samples (tooth) and postcranial long bones (femur and tibia). A total amount of 88 samples were selected from 38 different individuals. The samples were extracted by using an organic extraction protocol, DNA quantification by Quantifiler Trio kit and amplified with GlobalFiler kit. Results show that petrous bone outperforms other bone remains in quantification data, yielding 15–30 times more DNA than the others. DNA profile data presented likeness between petrous bone and tooth regarding detected alleles; however, the amount of DNA extracted in petrous bones allowed us to obtain more informative DNA profiles with superior quality. In conclusion, petrous bone or teeth sampling is recommended if DNA typing is going to be performed with environmentally degraded skeletal remains.     

A study of molecular complex formation between propyl gallate and ascorbic acid in the microemulsion phase of sodium dodecyl sulfate, pentanol and water system

The association between two water-soluble antioxidants, i.e. ascorbic acid and propyl gallate have been studied by absorption spectroscopy in microemulsion formed in sodium dodecyl sulfate/pentanol/water micellar system. It has been shown that propyl gallate forms 1:1 molecular complex with ascorbic acid in every solution. Evolution of the absorption spectra during the study of molecular complex formation goes through well-defined isosbestic points. The association constants were calculated using curve-fitting procedure. The observed interactions are stronger in the less polar solvents.     

A Selective Voltammetric Method for Uric Acid Detection at a Glassy Carbon Electrode Modified with Electrodeposited Film Containing DNA and Pt‐Fe(III) Nanocomposites

A novel biosensor by electrochemical codeposited Pt‐Fe(III) nanocomposites and DNA film was constructed and applied to the detection of uric acid (UA) in the presence of high concentration of ascorbic acid (AA). Based on its strong catalytic activity toward the oxidation of UA and AA, the modified electrode resolved the overlapping voltammetric response of UA and AA into two well‐defined peaks with a large anodic peak difference (ΔE_pa) of about 380mV. The catalytic peak current obtained from differential pulse voltammetry (DPV) was linearly dependent on the UA concentration from 3.8×10^?6 to 1.6×10^?4 M (r=0.9967) with coexistence of 5.0×10^?4 M AA. The detection limit was 1.8×10^?6 M (S/N=3) and the presence of 20 times higher concentration of AA did not interfere with the determination. The modified electrode shows good sensitivity, selectivity and stability.     

A label-free, quadruplex-based functional molecular beacon (LFG4-MB) for fluorescence turn-on detection of DNA and nuclease

We demonstrate a novel concept for the construction of a label-free, quadruplex-based functional molecular beacon (LFG4-MB) by using G-quadruplex motif as a substitute for Watson-Crick base pairing in the MB stem and a specific G-quadruplex binder, N-methyl mesoporphyrin IX (NMM) as a reporter. It shows high sensitivity in assays for UDG activity/inhibition and detection of DNA sequence based on the unique fluorescence increase that occurs as a result of the strong interaction between NMM and the folded quadruplex upon removal of uracil by UDG or displacement of block sequence by target DNA. The LFG4-MB is simple in design, fast in operation and could be easily transposed to other biological relevant target analysis by simply changing the recognition portion. The LFG4-MB does not require any chemical modification for DNA, which offers the advantages of simplicity and cost efficiency and obviates the possible interference with the affinity and specificity of the MB as well as the kinetic behavior of the catalysts caused by the bulky fluorescent groups. More importantly, the LFG4-MB offers great extent of freedom to tune the experimental conditions for the general applicability in bioanalysis.     

Equilibrium investigation of complex formation reactions involving copper(II), nitrilo-tris(methyl phosphonic acid) and amino acids,peptides or DNA constitutents. The kinetics,mechanism and correlation of rates with complex stability for metal ion promoted hydrolysis of glycine methyl ester

The complex formation reactions of [Cu(NTP)(OH₂)]^4? (NTP?=?nitrilo-tris(methyl phosphonic acid)) with some selected bio-relevant ligands containing different functional groups, are investigated. Stoichiometry and stability constants for the complexes formed are reported. The results show that the ternary complexes are formed in a stepwise mechanism whereby NTP binds to copper(II), followed by coordination of amino acid, peptide or DNA. Copper(II) is found to form Cu(NTP)H _n species with n?=?0, 1, 2 or 3. The concentration distribution of the various complex species has been evaluated. The kinetics of base hydrolysis of glycine methyl ester in the presence of copper(II)-NTP complex is studied in aqueous solution at different temperatures. It is proposed that the catalysis of GlyOMe ester occurs by attack of OH^? ion on the uncoordinated carbonyl carbon atom of the ester group. Activation parameters for the base hydrolysis of the complex [Cu(NTP)NH₂CH₂CO₂Me]^4? are, ΔH^±?=?9.5?±?0.3?kJ?mol^?1 and ΔS^±?=??179.3?±?0.9?J?K^?1?mol^?1. These show that catalysis is due to a substantial lowering of ΔH^±.     

A Facile Semisynthesis and Evaluation of Garcinoic Acid and Its Analogs for the Inhibition of Human DNA Polymerase β

Garcinoic acid has been identified as an inhibitor of DNA polymerase β (pol β). However, no structure-activity relationship (SAR) studies of garcinoic acid as a pol β inhibitor have been conducted, in part due to the lack of an efficient synthetic method for this natural product and its analogs. We developed an efficient semi-synthetic method for garcinoic acid and its analogs by starting from natural product δ-tocotrienol. Our preliminary SAR studies provided a valuable insight into future discovery of garcinoic acid-based pol β inhibitors.     

A comparison study between a disposable electrochemical DNA biosensor and a Vibrio fischeri-based luminescent sensor for the detection of toxicants in water samples

In the present study, a comparison between a disposable electrochemical DNA biosensor and a Vibrio fischeri-based luminescent sensor for the detection of toxicants in water samples was made.In order to realize this study, a disposable electrochemical DNA biosensor has been reported. The DNA biosensor is assembled by immobilizing double stranded Calf Thymus DNA onto the surface of a disposable carbon screen-printed electrode. The oxidation signal of the guanine base, obtained by a square wave voltammetric scan, is used as analytical signal to detect the DNA damage; the presence of low molecular weight compounds with affinity for nucleic acids is measured by their effect on the guanine oxidation peak.Wastewater samples provided during First European Interlaboratory Exercise on water toxicity in the course of the project SWIFT-WFD were analyzed, and biosensor results were compared with a currently used toxicity test ToxAlert^®100 based on the bioluminescence inhibition of Vibrio fischeri. This test have been used because is rapid, easy handling and cost effectively responses for the toxicity assessment in real water samples.The results showed a promising correlation between two tests used for the detection of toxic compounds in water samples.     

A Photosensitized Singlet Oxygen (1O2) Toolbox for Bio-Organic Applications: Tailoring 1O2 Generation for DNA and Protein Labelling,Targeting and Biosensing

Singlet oxygen (¹O₂) is the excited state of ground, triplet state, molecular oxygen (O₂). Photosensitized ¹O₂ has been extensively studied as one of the reactive oxygen species (ROS), responsible for damage of cellular components (protein, DNA, lipids). On the other hand, its generation has been exploited in organic synthesis, as well as in photodynamic therapy for the treatment of various forms of cancer. The aim of this review is to highlight the versatility of ¹O_2, discussing the main bioorganic applications reported over the past decades, which rely on its production. After a brief introduction on the photosensitized production of ¹O₂, we will describe the main aspects involving the biologically relevant damage that can accompany an uncontrolled, aspecific generation of this ROS. We then discuss in more detail a series of biological applications featuring ¹O₂ generation, including protein and DNA labelling, cross-linking and biosensing. Finally, we will highlight the methodologies available to tailor ¹O₂ generation, in order to accomplish the proposed bioorganic transformations while avoiding, at the same time, collateral damage related to an untamed production of this reactive species.     

A rugged and transferable method for determining blood cadmium,mercury, and lead with inductively coupled plasma-mass spectrometry

A simple, high-throughput method for determining total cadmium, mercury, and lead in blood in cases of suspected exposure, using inductively coupled plasma-mass spectrometry (ICP-MS), has been developed and validated. One part matrix-matched standards, blanks, or aliquots of blood specimens were diluted with 49 parts of a solution containing 0.25% (w/w) tetramethylammonium hydroxide; 0.05% v/v Triton X-100 (blood cell membranes and protein solubilization); 0.01% (w/v) ammonium pyrolidinedithiocarbamate (mercury memory effect prevention and oxidation state stabilization, solubilization by complexation of all three metals); 1% v/v isopropanol (signal enhancement); and 10 μg/L iridium (internal standard). Thus the final dilution factor is 1 + 49. The method provides the basis for the determination of total cadmium, mercury, and lead for assessment of environmental, occupational, accidental ingestion or elevated exposures from other means. Approximately 80 specimens, including blanks, calibration standards, and quality control materials can be processed in an 8-h day. The method has been evaluated by examining reference materials from the National Institute of Standards and Technology, as well as by participation in six rounds of proficiency testing intercomparisons led by the Wadsworth Center of the New York State Department of Health. This method was developed for the purpose of increasing U.S. emergency response laboratory capacity. To this end, 33 U.S. state, and 1 district health department laboratories have validated this method in their own laboratories.     

Open tubular capillary electrochromatography: A useful microreactor for collagen I glycation and interaction studies with low-density lipoprotein particles

Diabetes, a multifunctional disease and a major cause of morbidity and mortality in the industrialized countries, strongly associates with the development and progression of atherosclerosis. One of the consequences of high level of glucose in the blood circulation is glycation of long-lived proteins, such as collagen I, the most abundant component of the extracellular matrix (ECM) in the arterial wall. Glycation is a long-lasting process that involves the reaction between a carbonyl group of the sugar and an amino group of the protein, usually a lysine residue. This reaction generates an Amadori product that may evolve in advanced glycation end products (AGEs). AGEs, as reactive molecules, can provoke cross-linking of collagen I fibrils. Since binding of low-density lipoproteins (LDLs) to the ECM of the inner layer of the arterial wall, the intima, has been implicated to be involved in the onset of the development of an atherosclerotic plaque, collagen modifications, which can affect the affinity of native and oxidized LDL for collagen I, can promote the entrapment of LDLs in the intima and accelerate the progression of atherosclerosis.In this study, open tubular capillary electrochromatography is proposed as a new microreactor to study in situ glycation of collagen I. The kinetics of glycation was first investigated in a fused silica collagen I-coated capillary. Dimethyl sulphoxide, injected as an electroosmotic flow marker, gave information about the charge of coating. Native and oxidized LDL, and selected peptide fragments from apolipoprotein B-100, the protein covering LDL particles, were injected as marker compounds to clarify the interactions between LDLs and the glycated collagen I coating. The method proposed is simple and inexpensive, since only small amounts of collagen and LDL are required. Atomic force microscopy images complemented our studies, highlighting the difference between unmodified and glycated collagen I surfaces.