Hydrolytic endonucleolytic ribozyme (HYER): Systematic identification, characterization and potential application in nucleic acid manipulation.

Over the past decades, many drugs based on the use of nanotechnology and nucleic acids have been developed. However, until recently, most of them remained at the stage of pre-clinical development and testing and did not find their way to the clinic. In our opinion, the main reason for this situation lies in the enormous complexity of the development and industrial production of such formulations leading to their high cost. The development of nanotechnology-based drugs requires the participation of scientists from many and completely different specialties including Pharmaceutical Sciences, Medicine, Engineering, Drug Delivery, Chemistry, Molecular Biology, Physiology and so on. Nevertheless, emergence of coronavirus and new vaccines based on nanotechnology has shown the high efficiency of this approach. Effective development of vaccines based on the use of nucleic acids and nanomedicine requires an understanding of a wide range of principles including mechanisms of immune responses, nucleic acid functions, nanotechnology and vaccinations. In this regard, the purpose of the current review is to recall the basic principles of the work of the immune system, vaccination, nanotechnology and drug delivery in terms of the development and production of vaccines based on both nanotechnology and the use of nucleic acids.

Kinetic Selectivity of Complementary Nucleic Acids: bcr-abl-directed Antisense RNA and Ribozymes

The application of molecular biological techniques to immunological studies is based in large part on the manipulation of nucleic acids--RNA, DNA, and oligonucleotides. This unit offers very basic procedures that are used in a number of protocols later in the chapter. In particular, preparation of nucleoside triphosphates--the substrates for almost all synthetic reactions using nucleic acids--is described; the relevant radioisotopes, (32)P, (35)S, and (3)H are discussed; and methods for quantitating radioisotope and biotin incorporation into nucleic acids are presented. Finally, column chromatographic (gel filtration) procedures for separating labeled DNA from synthetic precursors are described.

Chapter 5 - Manipulation of Nucleic Acids

<p>Anthropization is the transformation that human actions exert on the environment. Artificial interventions modify the morphology of the ground and affect physical and chemical properties of natural terrain. Therefore, providing information on the distribution of artificial ground throughout the territory is necessary for land management, development and sustainability. Despite the effects of anthropization, from a geological approach, the systematic characterization of anthropic ground on a regional scale is scarcely developed in Catalonia.</p><p>In the last decade, one of the lines of work of Institut Cartogràfic i Geològic de Catalunya (the Catalan geological survey organisation) has been the development of the project Geoanthropic map of Catalonia, which incorporate information of active geological processes and artificial ground. Up to now, the activity in this project has broadly consisted of publishing several map sheets of 1:25.000 scale from different areas of Catalonia (5.000 km<sup>2</sup> from 32.108,2 km<sup>2</sup>). Recently, in the framework of this project, it is proposed to refocus with the purpose of ​​providing information on these two themes from all over the territory. In this process, in relation to artificial interventions, an analysis has been carried out to determine which anthropic terrains and related information can be obtained for its usefulness in a systematic way in the medium term.</p><p>In this analysis, firstly, the available reference information sources have been established from which information on anthropic lands in Catalonia can be extracted. Basically, these documents are topographic maps, geothematic maps, land use map, digital elevation models and other historical cartographic documents. Much of the existing information in these sources must be redirected to a more geological approach so that it can be used to address aspects related to geotechnics, natural hazards, soil pollution and other environmental concerns.</p><p>Secondly, based on data analysis, a series of certain anthropic lands have been evaluated which can be captured on a systematic identification at regional scale. Thereby, the following anthropogenic terrains have been established: built-up areas, agricultural areas, sealed ground, urban compacity, worked grounds (e.g., related to mineral excavations and transport infrastructures), engineered embankments, infilled excavations and other more singular anthropogenic deposits. Therefore, from a geological perspective, it will be feasible to identify and map these anthropic lands and provide this information throughout the Catalan territory in the medium term.</p><p>Bearing in mind all the above, the presentation will consist of this general analysis and the considerations that have been extracted regarding this. In addition, the preliminary results of the systematically characterized artificial ground will be shown.</p>

The use of nucleic acids to estimate crustacean growth is not well documented, and may be complicated by biochemical changes associated with their molt cycle. The objectives of this study were to assess the effects of molt stage on nucleic acid concentrations in abdominal muscle tissue of juvenile white shrimp,Penaeus vannamei, and to examine the relationship between nucleic acid concentrations and growth rates of shrimp exposed to different feeding regimes throughout a 12 d feeding experiment. RNA and DNA concentrations and RNA:DNA ratios were not significantly different among the five major molt stages early postmolt, late postmolt, intermolt, early premolt, and late premolt. In the feeding experiment, RNA concentrations and RNA:DNA ratios accounted for >70% of the variation in shrimp growth on three different sampling days. In addition, RNA concentrations and RNA:DNA ratios were used successfully to discriminate between unfed, moderately-fed, and well-fed shrimp. These variables exhibited significant treatment differences in <24 h after the initiation of the different feeding regimes, whereas significant changes in whole-body weight took longer to detect. Rapid detection of significant treatment effects can be useful in ecological studes, especially those concerned with food-web interactions.

Chapter 5 - Manipulation of Nucleic Acids

Chapter e5 - Manipulation of Nucleic Acids

The research for new products against pathogens, parasites and infesting species, in both agriculture and medicine, implies huge and increasing scientific, industrial and economic efforts. Traditional approaches are based on random screening procedures searching for bioactive compounds. However, the success of such methodologies in most cases has been strongly limited by side-effects of the potential new drugs, especially toxicity and pharmacological resistance. The use of nucleic acids in drug development has been introduced searching for target-specific effect. In addition, a recent discovery revealed that randomly fragmented extracellular self-DNA may act as highly species-specific inhibitory product for different species, suggesting an unprecedented use of DNA for biological control. On this base, a new scenario of pharmacological applications is discussed.

RNA editing modifies the sequence of primary transcripts, potentially resulting in profound effects to RNA structure and protein-coding sequence. Recent analyses of RNA sequence data are beginning to provide insights into the distribution of RNA editing across the entire transcriptome, but there are few published matched whole genome and transcriptome sequence datasets, and designing accurate bioinformatics methodology has proven highly challenging. To further characterize the RNA editome, we analyzed 16 paired DNA-RNA sequence libraries from prostate tumor specimens, employing a comprehensive strategy to rescue low coverage sites and minimize false positives. We identified over a hundred thousand putative RNA editing events, a third of which were recurrent in two or more samples, and systematically characterized their type and distribution across the genome. Within genes the majority of events affect non-coding regions such as introns and untranslated regions (UTRs), but 546 genes had RNA editing events predicted to result in deleterious amino acid alterations. Finally, we report a potential association between RNA editing of microRNA binding sites within 3′ UTRs and increased transcript expression. These results provide a systematic characterization of the landscape of RNA editing in low coverage sequence data from prostate tumor specimens. We demonstrate further evidence for RNA editing as an important regulatory mechanism and suggest that the RNA editome should be further studied in cancer.

Long non-coding RNA (lncRNA) structurally resembles mRNA but cannot be translated into protein. Although the systematic identification and characterization of lncRNAs have been increasingly reported in model species, information concerning non-model species is still lacking. Here, we report the first systematic identification and characterization of lncRNAs in two sea cucumber species: (1) Apostichopus japonicus during lipopolysaccharide (LPS) challenge and in heathy tissues and (2) Holothuria glaberrima during radial organ complex regeneration, using RNA-seq datasets and bioinformatics analysis. We identified A. japonicus and H. glaberrima lncRNAs that were differentially expressed during LPS challenge and radial organ complex regeneration, respectively. Notably, the predicted lncRNA-microRNA-gene trinities revealed that, in addition to targeting protein-coding transcripts, miRNAs might also target lncRNAs, thereby participating in a potential novel layer of regulatory interactions among non-coding RNA classes in echinoderms. Furthermore, the constructed coding-non-coding network implied the potential involvement of lncRNA-gene interactions during the regulation of several important genes (e.g., Toll-like receptor 1 [TLR1] and transglutaminase-1 [TGM1]) in response to LPS challenge and radial organ complex regeneration in sea cucumbers. Overall, this pioneer systematic identification, annotation, and characterization of lncRNAs in echinoderm pave the way for similar studies and future genetic, genomic, and evolutionary research in non-model species.

Proteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks. For the screening of valosin-containing protein, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners, we utilized a membrane-based array technology that allows the identification of human protein-protein interactions with crude bacterial cell extracts. Many novel interaction pairs such as valosin-containing protein/autocrine motility factor receptor, CHIP/caytaxin, or amphiphysin II/DLP4 were identified and subsequently confirmed by pull-down, two-hybrid and co-immunoprecipitation experiments. In addition, assays were performed to validate the interactions functionally. CHIP e.g. was found to efficiently polyubiquitinate caytaxin in vitro, suggesting that it might influence caytaxin degradation in vivo. Using peptide arrays, we also identified the binding motifs in the proteins DLP4, XRCC4, and fructose-1,6-bisphosphatase, which are crucial for the association with the Src homology 3 domain of amphiphysin II. Together these studies indicate that our human proteome array technology permits the identification of protein-protein interactions that are functionally involved in neurodegenerative disease processes, the degradation of protein substrates, and the transport of membrane vesicles.

Extensive research endeavors have been initiated to explore the potential of nucleic acids, including siRNA, miRNA, catalytic RNA (ribozymes), aptamer RNA (RNA with exquisite roles similar to protein receptors) and antisense oligonucleotides, as therapeutic agents. Some antisense oligonucleotides and aptamer RNA have reached clinical applications, while significant amounts of clinical trials for siRNA are underway. Direct use of nucleic acids in medicine, however, faces serious hurdles such as poor cell specificity and uptake of nucleic acids, and inaccessibility of nucleic acids to cell nuclei. These uptake problems of nucleic acids are mainly ascribed to inefficiency of nucleic acids to permeate biological barriers after administration. Using peptides as vectors (ligands) to assist nucleic acid trafficking across the plasma membrane are rational strategies to develop nucleic acid-based therapeutic reagents. In the current study, we optimized previously developed aqueous phase two-step nucleic acid phosphoramidation to facilitate synthesis of peptide-oligonucleotide conjugates (POCs). We improved POC yields by exploiting 4(5)-methylimidazole, specific surfactants with defined concentrations and diamine derivatized peptides. Optimized aqueous phase two-step nucleic acid phosphoramidation was applied to efficiently conjugate peptides with oligonucleotides, with or without a disulfide-containing linker, to generate corresponding POCs with yields of 47-75%. Moreover, we exploited the optimized two-step nucleic acid phosphoramidation to developing a novel, simpler and more effective labeling method for conjugating nucleic acids with fluorophores. Studied by flow cytometry and laser scanning confocal microscope, we unequivocally demonstrated FITC-labeled POCs were taken up by A549 cells effectively. However, when employing the methods to synthesize POCs for developing nucleic acid drugs to inhibit the growth of E. coli, our results indicated that the tailored-made POCs did not meet our expectation of conferring antibacterial and antibiotic effects. In summary, this study successfully optimized previously developed aqueous phase two-step nucleic acid phosphoramidation to facilitate POC synthesis. We also demonstrated that those peptides in POCs served the purpose of Trojan horses to deliver nucleic acids into the mammalian cells. The outcomes of the research shed light on the mechanisms of aqueous-phase phosphoramidation reactions and, moreover, provide the essential supports for POCs to develop effective therapeutic reagents in clinics to treat human diseases.

Long intergenic non-coding RNAs (lincRNAs) play important roles in many cellular processes. Here, we present the first systematic identification and characterization of lincRNAs in fetal porcine skeletal muscle. We obtained a total of 55.02 million 90-bp paired-end reads and assembled 54,550 transcripts using cufflinks. We developed a pipeline to identify 570 multi-exon lincRNAs by integrating a set of previous approaches. These putative porcine lincRNAs share many characteristics with mammalian lincRNAs, such as a relatively short length, small number of exons and low level of sequence conservation. We found that the porcine lincRNAs were preferentially located near genes mediating transcriptional regulation rather than those with developmental functions. We further experimentally analyzed the features of a conserved mouse lincRNA gene and found that isoforms 1 and 4 of this lincRNA were enriched in the cell nucleus and were associated with polycomb repressive complex 2 (PRC2). Our results provide a catalog of fetal porcine lincRNAs for further experimental investigation of the functions of these genes in the skeletal muscle developmental process.

The use of nucleic acids (NAs) has revolutionized medical approaches and ushered in a new era of combating various diseases. Accordingly, there is an increasing demand for accurate identification, localization, quantification, and characterization of NAs encapsulated in nonviral or viral vectors. The vast spectrum of molecular dimensions and intra- and intermolecular interactions presents a formidable obstacle for NA analytical development. Typically, the comprehensive analysis of encapsulated NAs, free NAs, and their spatial distribution poses a challenge that is seldom tackled in its complete complexity. The identification of appropriate physicochemical methodologies for large nonencapsulated or encapsulated NAs is particularly intricate and necessitates an evaluation of the analytical outcomes and their appropriateness in addressing critical quality attributes. In this work, we examine the analytics of non-encapsulated or encapsulated large NAs(>500 nucleotides) utilizing capillary electrophoresis (CE) and liquid chromatography (LC) methodologies such as free zone CE, gel CE, affinity CE, and ion pair high-performance liquid chromatography (HPLC). These methodologies create a complete picture of the NA's critical quality attributes, including quantity, identity, purity, and content ratio.