Mechanisms of base selection by human single-stranded selective monofunctional uracil-DNA glycosylase

hSMUG1 (human single-stranded selective monofunctional uracil-DNA glyscosylase) is one of three glycosylases encoded within a small region of human chromosome 12. Those three glycosylases, UNG (uracil-DNA glycosylase), TDG (thymine-DNA glyscosylase), and hSMUG1, have in common the capacity to remove uracil from DNA. However, these glycosylases also repair other lesions and have distinct substrate preferences, indicating that they have potentially redundant but not overlapping physiological roles. The mechanisms by which these glycosylases locate and selectively remove target lesions are not well understood. In addition to uracil, hSMUG1 has been shown to remove some oxidized pyrimidines, suggesting a role in the repair of DNA oxidation damage. In this paper, we describe experiments in which a series of oligonucleotides containing purine and pyrimidine analogs have been used to probe mechanisms by which hSMUG1 distinguishes potential substrates. Our results indicate that the preference of hSMUG1 for mispaired uracil over uracil paired with adenine is best explained by the reduced stability of a duplex containing a mispair, consistent with previous reports with Escherichia coli mispaired uracil-DNA glycosylase. We have also extended the substrate range of hSMUG1 to include 5-carboxyuracil, the last in the series of damage products from thymine methyl group oxidation. The properties used by hSMUG1 to select damaged pyrimidines include the size and free energy of solvation of the 5-substituent but not electronic inductive properties. The observed distinct mechanisms of base selection demonstrated for members of the uracil glycosylase family help explain how considerable diversity in chemical lesion repair can be achieved

An ultrasensitive photoelectrochemical nucleic acid biosensor

A simple and ultrasensitive procedure for non-labeling detection of nucleic acids is described in this study. It is based on the photoelectrochemical detection of target nucleic acids by forming a nucleic acid/photoreporter adduct layer on an ITO electrode. The target nucleic acids were hybridized with immobilized oligonucleotide capture probes on the ITO electrode. A subsequent binding of a photoreporter—a photoactive threading bis-intercalator consisting of two N,N′-bis(3-propyl-imidazole)-1,4,5,8-naphthalene diimides (PIND) linked by a [Formula: see text] (bpy = 2,2′-bipyridine) complex (PIND–Ru–PIND)—allowed for photoelectrochemical detection of the target nucleic acids. The extremely low dissociation rate of the adduct and the highly reversible photoelectrochemical response under visible light illumination (490 nm) make it possible to conduct nucleic acid detection, with a sensitivity enhancement of four orders of magnitude over voltammetry. These results demonstrate for the first time the potential of photoelectrochemical biosensors for PCR-free ultrasensitive detection of nucleic acids

Colloquium: Physical approaches to DNA sequencing and detection

With the continued improvement of sequencing technologies, the prospect of genome-based medicine is now at the forefront of scientific research. To realize this potential, however, a revolutionary sequencing method is needed for the cost-effective and rapid interrogation of individual genomes. This capability is likely to be provided by a physical approach to probing DNA at the single-nucleotide level. This is in sharp contrast to current techniques and instruments that probe (through chemical elongation, electrophoresis, and optical detection) length differences and terminating bases of strands of DNA. Several physical approaches to DNA detection have the potential to deliver fast and low-cost sequencing. Central to these approaches is the concept of nanochannels or nanopores, which allow for the spatial confinement of DNA molecules. In addition to their possible impact in medicine and biology, the methods offer ideal test beds to study open scientific issues and challenges in the relatively unexplored area at the interface between solids, liquids, and biomolecules at the nanometer length scale. This Colloquium emphasizes the physics behind these methods and ideas, critically describes their advantages and drawbacks, and discusses future research opportunities in the field

Bioinorganic Chemistry

This book covers material that could be included in a one-quarter or one-semester course in bioinorganic chemistry for graduate students and advanced undergraduate students in chemistry or biochemistry. We believe that such a course should provide students with the background required to follow the research literature in the field. The topics were chosen to represent those areas of bioinorganic chemistry that are mature enough for textbook presentation. Although each chapter presents material at a more advanced level than that of bioinorganic textbooks published previously, the chapters are not specialized review articles. What we have attempted to do in each chapter is to teach the underlying principles of bioinorganic chemistry as well as outlining the state of knowledge in selected areas. We have chosen not to include abbreviated summaries of the inorganic chemistry, biochemistry, and spectroscopy that students may need as background in order to master the material presented. We instead assume that the instructor using this book will assign reading from relevant sources that is appropriate to the background of the students taking the course. For the convenience of the instructors, students, and other readers of this book, we have included an appendix that lists references to reviews of the research literature that we have found to be particularly useful in our courses on bioinorganic chemistry

Mechanisms for DNA Charge Transport

DNA charge transport (CT) chemistry has received considerable attention by scientific researchers over the past 15 years since our first provocative publication on long range CT in a DNA assembly.1,2 This interest, shared by physicists, chemists and biologists, reflects the potential of DNA CT to provide a sensitive route for signaling, whether in the construction of nanoscale biosensors or as an enzymatic tool to detect damage in the genome. Research into DNA CT chemistry began as a quest to determine whether the DNA double helix, a macromolecular assembly in solution with π-stacked base pairs, might share conductive characteristics with π-stacked solids. Physicists carried out sophisticated experiments to measure the conductivity of DNA samples, but the means to connect discrete DNA assemblies into the devices to gauge conductivity varied, as did the conditions under which conductivities were determined. Chemists constructed DNA assemblies to measure hole and electron transport in solution using a variety of hole and electron donors. Here, too, DNA CT was seen to depend upon the connections, or coupling, between donors and the DNA base pair stack. Importantly, these experiments have resolved the debate over whether DNA CT is possible. Moreover these studies have shown that DNA CT, irrespective of the oxidant or reductant used to initiate the chemistry, can occur over long molecular distances but can be exquisitely sensitive to perturbations in the base pair stack. Here we review some of the critical characteristics of DNA charge transport chemistry, taking examples from a range of systems, and consider these characteristics in the context of their mechanistic implications. This review is not intended to be exhaustive but instead to be illustrative. For instance, we describe studies involving measurements in solution using pendant photooxidants to inject holes, conductivity studies with covalently modified assemblies, and electrochemical studies on DNA-modified electrodes. We do not focus in detail on the differences amongst these constructs but instead on their similarities. It is the similarity among these various systems that allows us to consider different mechanisms to describe DNA CT. Thus we review also the various mechanisms for DNA CT that have been put forth and attempt to reconcile these mechanistic proposals with the many disparate measurements of DNA CT. Certainly the debate among researchers has shifted from "is DNA CT possible?" to "how does it work?". This review intends to explore this latter question in detail

Role of tautomerism in RNA biochemistry

Heterocyclic nucleic acid bases and their analogs can adopt multiple tautomeric forms due to the presence of multiple solvent-exchangeable protons. In DNA, spontaneous formation of minor tautomers has been speculated to contribute to mutagenic mispairings during DNA replication, whereas in RNA, minor tautomeric forms have been proposed to enhance the structural and functional diversity of RNA enzymes and aptamers. This review summarizes the role of tautomerism in RNA biochemistry, specifically focusing on the role of tautomerism in catalysis of small self-cleaving ribozymes and recognition of ligand analogs by riboswitches. Considering that the presence of multiple tautomers of nucleic acid bases is a rare occurrence, and that tautomers typically interconvert on a fast time scale, methods for studying rapid tautomerism in the context of nucleic acids under biologically relevant aqueous conditions are also discussed.National Institutes of Health (U.S.) (Grant P01 CA26731)National Institutes of Health (U.S.) (Grant R37 CA080024)National Institutes of Health (U.S.) (Grant P30 ES002109)National Institutes of Health (U.S.) (Training Grant T32 ES007020

Design and Syntheses of Novel Quenchers for Fluorescent Hybridization Probes

Since most of human diseases are related to genetic mutations, during the past two decades, identification of such mutations has attracted much attention. Detection of these mutations is mainly based hybridization with the complementary reporter probes. Nucleic acids detection takes place by changing either the reporter’s fluorescence intensity or the colour of its fluorescence. The use of fluorescent probes for nucleic acid detection has attracted much attention due to its efficiency, the ease of synthesis and availability of commercial reporters that facilitates the probe synthesis. Nowadays, most of nucleic acid detection using fluorescent probes relies on quenching of fluorescence by energy transfer from one fluorophore to another or to nonfluorescent molecule (quencher). The most common, widely used, quencher in fluorescent probes is 4-(N,N-dimethylamino)azobenzene-4\u27-carboxylic acid (DABCYL). The goal of this thesis was to introduce new quenchers which structurally mimic the universal quencher DABCYL into peptide nucleic acid (PNA), DNA and RNA probes. These quenchers are also characterized by their ability to undergo photoisomerization upon illumination with light

Non-Natural Nucleotides As Probes For The Mechanism And Fidelity Of DNA Polymerases

DNA is a remarkable macromolecule that functions primarily as the carrier of the genetic information of organisms ranging from viruses to bacteria to eukaryotes. The ability of DNA polymerases to efficiently and accurately replicate genetic material represents one of the most fundamental yet complex biological processes found in nature. The central dogma of DNA polymerization is that the efficiency and fidelity of this biological process is dependent upon proper hydrogen-bonding interactions between an incoming nucleotide and its templating partner. However, the foundation of this dogma has been recently challenged by the demonstration that DNA polymerases can effectively and, in some cases, selectively incorporate non-natural nucleotides lacking classic hydrogen-bonding capabilities into DNA. In this review, we describe the results of several laboratories that have employed a variety of non-natural nucleotide analogs to decipher the molecular mechanism of DNA polymerization. The use of various non-natural nucleotides has lead to the development of several different models that can explain how efficient DNA synthesis can occur in the absence of hydrogen-bonding interactions. These models include the influence of steric fit and shape complementarity, hydrophobicity and solvation energies, base-stacking capabilities, and negative selection as alternatives to rules invoking simple recognition of hydrogen-bonding patterns. Discussions are also provided regarding how the kinetics of primer extension and exonuclease proofreading activities associated with high-fidelity DNA polymerases are influenced by the absence of hydrogen-bonding functional groups exhibited by non-natural nucleotides

Localization of metal ions in DNA

M-DNA is a novel complex formed between DNA and transition metal ions under alkaline conditions.  The unique properties of M-DNA were manipulated in order to rationally place metal ions at specific regions within a double-stranded DNA helix.   Investigations using thermal denaturation profiles and the ethidium fluorescence assay illustrate that the pH at which M-DNA formation occurs is influenced heavily by the DNA sequence and base composition.  For instance, DNA with a sequence consisting of poly[d(TG)•d(CA)] is completely converted to M-DNA at pH 7.9 while DNA consisting entirely of poly[d(AT)] remains in the B-DNA conformation until a pH of 8.6 is reached.  The pH at which M-DNA formation occurs is further decreased by the incorporation of 4-thiothymine (s4T).  DNA oligomers with a mixed sequence composed of half d(AT) and the other half d(TG)•d(CA) showed that only 50% of the DNA is able to incorporate Zn2+ ions at pH 7.9.  This suggests that only regions corresponding to the tracts of d(TG)•d(CA) are being transformed.   Duplex DNA monolayers were self-assembled on gold through a Au-S linkage and both B- and M-DNA conformations were studied using X-ray photoelectron spectroscopy (XPS) in order to better elucidate the location of the metal ions.  The film thickness, density, elemental composition and ratios for samples were analyzed and compared.  The DNA surface coverage, calculated from both XPS and electrochemical measurements, was approximately 1.2 x 1013 molecules/cm2 for B-DNA.  All samples showed distinct peaks for C 1s, O 1s, N 1s, P 2p and S 2p as expected for a thiol-linked DNA.  On addition of Zn2+ to form M-DNA the C 1s, P 2p and S 2p showed only small changes while both the N 1s and O 1s spectra changed considerably.  This result is consistent with Zn2+ interacting with oxygen on the phosphate backbone as well as replacing the imino protons of thymine (T) and guanine (G) in M-DNA.   Analysis of the Zn 2p spectra also demonstrated that the concentration of Zn2+ present under M-DNA conditions is consistent with Zn2+ binding to both the phosphate backbone as well as replacing the imino protons of T or G in each base pair.  After the M-DNA monolayer is washed with a buffer containing only Na+ the Zn2+ bound to the phosphate backbone is removed while the Zn2+ bound internally still remains. Variable angle x-ray photoelectron spectroscopy (VAXPS) was also used to examine monolayers consisting of mixed sequence oligomers.  Preliminary results suggest that under M-DNA conditions, the zinc to phosphate ratio changes relative to the position of the d(TG)•d(CA) tract being at the top or bottom of the monolayer.    Electrochemistry was also used to investigate the properties of M-DNA monolayers on gold and examine how the localization of metal ions affects the resistance through the DNA monolayer.  The effectiveness of using the IrCl62-/3- redox couple to investigate DNA monolayers and the potential advantages of this system over the standard Fe(CN)63-/4- redox couple are demonstrated.  B-DNA monolayers were converted to M-DNA by incubation in buffer containing 0.4 mM Zn2+ at pH 8.6 and studied by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronoamperometry (CA) with IrCl62-/3-.   Compared to B-DNA, M-DNA showed significant changes in CV, EIS and CA spectra.  However, only small changes were observed when the monolayers were incubated in Mg2+ at pH 8.6 or in Zn2+ at pH 6.0.  The heterogeneous electron-transfer rate (kET) between the redox probe and the surface of a bare gold electrode was determined to be 5.7 x 10-3 cm/s.  For a B-DNA modified electrode, the kET through the monolayer was too slow to be measured.  However, under M-DNA conditions, a kET of 1.5 x 10-3 cm/s was reached.  As well, the percent change in resistance to charge transfer (RCT), measured by EIS, was used to illustrate the dependence of M-DNA formation on pH.  This result is consistent with Zn2+ ions replacing the imino protons on thymine and guanine residues.  Also, at low pH values, the percent change in RCT seems to be greater for d(TG)15•d(CA)15 compared to oligomers with mixed d(AT) and d(TG)•d(CA) tracts.  The IrCl62-/3- redox couple was also effective in differentiating between single-stranded and double-stranded DNA during dehybridization and rehybridization experiments.