Publisher Summary This chapter discusses the progress and techniques of RNA structure and modeling. To understand the structure of RNA, it is necessary to first understand some of the unique biochemical properties associated with RNA and its structural elements. These properties directly lead to the formation of structural elements and motifs in tertiary structures. These include not only basic structure elements like helices, loops, bulges, and junctions but also less common elements like ribose zipper and tetraloop-receptor that contribute to the difficulty of tertiary structure prediction. The study of RNA structure started later than protein structure research, people followed the path of protein studies for experience and methods, such as crystallography and molecular dynamic simulation. A group of RNA molecules that have been used for structural studies are the ribozymes. A ribozyme is an RNA molecule that has the ability to catalyze a reaction. The characterization of RNA structure and structural dynamics is greatly enhanced by the application of chemical and enzymatic probes of RNA structure in solution. These probes enable to study RNA structures in conditions that are physiologically relevant and to investigate their interactions with partner molecules and to detect enzymatic active sites.

This chapter presents a broader view of the state of human disease modeling in Drosophila melanogaster and discusses new directions in the study of the genetic basis of human disorders in flies. The Drosophila classical genetics powerhouse, in combination with rapidly developing genomic and postgenomic tools, accelerates the identification and characterization of gene networks. Because the molecular mechanisms controlling a variety of physiological pathways are largely conserved between flies and humans, flies are quite useful in modeling a variety of human diseases. These include nervous system disorders, cancer, immune responses, elements of the cardiovascular system, and many more. Possibly the most successful area of human neurological disease modeling in Drosophila is the models of polyglutamine tract repeat disorders. The fly eye is an excellent readout for polyglutamine tract repeat disorders, like such as Huntington's disease and the spinocerebellar ataxias. In both conditions, there is a critical threshold of polyglutamine repeats that must be reached before a clinical presentation is observed. In flies, the expression of the human Huntingtin protein or the SCA3/MJD protein, containing the clinically relevant number of repeats, leads to the degeneration of photoreceptor neurons.

Publisher Summary This chapter discusses the molecular computing with deoxyribozymes. Silicomimetic molecular computing is based on an idea that individual molecules can perform basic logical operations and make simple decisions based on the presence or absence of multiple factors in solution. Using deoxyribozymes, nucleic acid catalysts made of DNA, and recognition regions for oligonucleotides—stem-loops—a so-called “full set of molecular logic gates,” was constructed in the study described in the chapter, which allowed for the combination of individual gates into more complex circuits. These gates and their circuits analyze the sets of oligonucleotides as inputs and produce changed substrate oligonucleotides as outputs. The chapter describes the initial efforts to integrate molecular computing devices with more traditional approaches to nanomedicine, such as those using nanoparticles for drug-delivery. This approach opens possibilities to increase the functional complexity of delivery systems. A three-layer cascade is described, in which microscopic particles coordinate their activity without any direct physical contact. The elementary unit of a network of microparticles is a single particle covered with a DNA computing or sensing element. Individual bead senses the presence of an input stimulus (or multiple stimuli) in solution, and, according to a set of rules encoded on this bead by computing elements, it releases an oligonucleotide signal as an output through a catalytic process.

Publisher Summary DNA polymerase epsilon (Pol epsilon) is a large, multi-subunit polymerase that is conserved throughout all eukaryotes. In addition to its role as one of the three DNA polymerases responsible for bulk chromosomal replication, Pol epsilon is implicated in a wide variety of important cellular processes, including the repair of damaged DNA, DNA recombination, and the regulation of proper cell cycle progression. Pol e catalyzes DNA template-dependent DNA synthesis by a phosphoryl transfer reaction involving nucleophilic attack by the 30 hydroxyl of the primer terminus on the a-phosphate of the incoming deoxynucleoside triphosphate (dNTP). The products of this reaction are pyrophosphate and a DNA chain increased in length by one nucleotide. The catalytic mechanism is conserved among DNA polymerases. It begins with binding of a primer template to the polymerase. Like all polymerases, Pol e ultimately does not generate all types of errors at equal rates, but rather has distinctive error specificity. Two features of Pol e error specificity are particularly interesting in light of its proposed biological roles in DNA replication. One is that Pol e is among the most accurate of DNA polymerases for single base deletion/insertion errors. Because indels are typically generated more frequently within repetitive sequences, this property may be relevant to the proposal that Pol e has a particularly important role in replicating heterochromatic DNA, which is enriched in repetitive sequences. Another is that a mutant derivative of Pol e has a unique base substitution error specificity that has been useful for inferring its role in replication of the leading strand template.

Publisher Summary This chapter provides an overview of the fluorescence correlation spectroscopy (FCS) technique, and focuses on its applications to the investigation of conformational dynamics of nucleic acids. FCS is a technique based on the measurement of the spontaneous fluctuations of the fluorescence signal of a small number of molecules. Fluorescence fluctuations are typically measured in an optically restricted submicron observation volume and then analyzed statistically to reveal kinetic information about the processes that lead to these fluctuations. Such processes include concentration fluctuations via molecular diffusion, chemical reactions, photophysical processes, and so on. To obtain dynamic or kinetic information from FCS experiments, the experimentally acquired auto- and cross-correlation functions have to be compared with the decays predicted by the appropriate physical models. The simplest FCS experiment involves measuring the intensity fluctuations of a fluorescent particle diffusing freely in three dimensions. For FCS to be useful in the study of conformational dynamics, the intensity of fluorescence of the probe has to be influenced by the conformation of the biopolymer being studied. The most common approach is to label the biopolymer with a fluorophore–quencher or donor–acceptor FRET pair, so that the efficiency of emission of the fluorophore is a direct measure of the distance between the two tags.

Publisher Summary This chapter discusses various scientific and practical applications of molecular colony technique (MCT) developed to date in different laboratories in Russia, United States, and China. In addition to already mentioned applications, these include in vitro gene cloning and molecular diagnostics. MCT comprises the amplification of nucleic acids in immobilized media, such as in a gel. Because the gel matrix prevents the convection of the entrapped liquid and restricts diffusion of nucleic acids, the amplification products do not spread throughout the medium. Rather, they form more or less compact molecular colonies, which are reminiscent of bacterial colonies growing on the surface of a nutrient agar. If amplification is carried out in a thin gel layer, a 2-D pattern of molecular colonies is generated, each colony comprising many copies (a clone) of a single starting RNA or DNA template molecule. The ability of MCT to detect single molecules makes it a unique tool for studying extremely rare reactions, such as recombinations between RNA molecules. After the discovery of genetic recombination, at the level of DNA, indications began to accumulate that a similar process may occur at the level of RNA as well.

Publisher Summary This chapter summarizes the current site-directed spin labeling (SDSL) studies on nucleic acids, with discussions focusing on literature from the last decade. SDSL is useful in studying high molecular weight systems under physiological conditions. It has been particularly successful in studying systems (e.g., membrane proteins) that are difficult to investigate using other methods, such as X-ray crystallography and NMR spectroscopy. SDSL has been used to study nucleic acids, and data suggest that one can obtain unique structural and dynamic information about DNA and RNA at the level of individual nucleotides. The majority of nucleic acid SDSL studies have used one of two types of EPR measurements. Distance measurements between pairs of nitroxides provide direct structural constraints in nucleic acid systems. In addition, the mobility of a single-labeled nitroxide can be measured to yield structural and dynamic information at the labeling site. Because nucleic acids are different from proteins in the nature of the basic chemical constituents (4 nucleotides vs. 20 amino acids) and their secondary structural units (B-form/A-form doubled-stranded helix vs. α -helix/ β -sheet), SDSL of nucleic acids requires unique methodologies, particularly in the areas of nitroxide attachment and the correlation of the nitroxide behavior to that of the parent molecule.

This chapter discusses the use of inhibitors of tyrosyl-DNA phosphodiesterase (Tdp1) and Chk1/2 in combination with Topoisomerase I (TopI) inhibitors. TopI is an abundant and essential enzyme. It is the selective target of camptothecins, which are effective anticancer agents. TopI–DNA cleavage complexes can also be trapped by various endogenous and exogenous DNA lesions, including mismatches, abasic sites, and carcinogenic adducts. Tdp1 is one of the repair enzymes for Top1–DNA covalent complexes. It forms a multiprotein complex that includes poly (Adenosine diphosphate (ADP)–ribose) polymerase (PARP). PARP-deficient cells are hypersensitive to camptothecins and functionally deficient for Tdp1. This chapter reviews the developments in several pathways involved in the repair of Top1 cleavage complexes and the role of Chk1 and Chk2 checkpoint kinases in the cellular responses to Top1 inhibitors. The genes conferring camptothecin hypersensitivity are compiled for humans, budding yeast, and fission yeast.

Publisher Summary This chapter provides a detailed summary about human Natural killer (NK) cells and their sophisticated competencies to discriminate between autologous healthy cells and autologous unhealthy cells that have been rendered ‘‘unusual’’ by malignant transformation, virus infection, or other means. In this chapter, heterogeneity of the NK cell population is a major topic, because this population heterogeneity provides the basis of broad specificity. This heterogeneity results mainly from a highly variable and at least in part stochastic expression of numerous inhibitory and eventually also activating receptors at the cell surface, and the surface expression of these receptors and the subsequent signal integration process are part of a regulatory network. The fine tuning of this network, probably by a nontarget-specific educational process, provides not only the basis for self-tolerance of NK cells to normal tissues but also allows fast and sensitive detection of the unusual properties of diseased cells, leading subsequently to their elimination.