Deoxyribozymes: useful DNA catalysts in vitro and in vivo

Deoxyribozymes (DNA enzymes; DNAzymes) are catalytic DNA sequences. Using the technique of in vitro selection, individual deoxyribozymes have been identified that catalyze RNA cleavage, RNA ligation, and a growing range of other chemical reactions. DNA enzymes have been used in vitro for applications such as biochemical RNA manipulation and analytical assays for metal ions, small organic compounds, oligonucleotides, and proteins. Deoxyribozymes have also been utilized as in vivo therapeutic agents to destroy specific mRNA targets. Although many conceptual and practical challenges remain to be addressed, deoxyribozymes have substantial promise to contribute meaningfully for applications both in vitro and in vivo.     

Peptide-mediated cellular delivery of antisense oligonucleotides and their analogues

Improving the delivery of synthetic oligonucleotides and their analogues into cells is an important goal in the full development of antisense technology for control of gene expression in cell culture and in vivo. This review describes the harnessing of certain peptides, either as noncovalent complexes or as covalent conjugates, to enhance the delivery of antisense oligonucleotides into cells and/or to affect their cell localization. Phosphodiester and phosphorothioate oligonucleotides are included as well as peptide nucleic acids (PNAs), analogues of oligonucleotides where the negatively charged phosphate backbone is replaced by a neutral amide linkage. This review contains a critical evaluation of claims for certain peptide-oligonucleotide conjugates to translocate into cultured cells by a non-energy-dependent nonendosomal route. In addition, the available evidence for the utility of stable versus nonstable linkages between peptide and oligonucleotide or PNA is discussed.     

Dps-like proteins: structural and functional insights into a versatile protein family

Dps-like proteins are key factors involved in the protection of prokaryotic cells from oxidative damage. They act by either oxidizing iron to prevent the formation of oxidative radicals or by forming Dps-DNA complexes to physically protect DNA. All Dps-like proteins are characterized by a common three-dimensional architecture and are found as spherical dodecamers with a hollow central cavity. Despite their structural similarities, recent biochemical and structural data have suggested different functions among members of the family that range from protection inside the cells in response to various stress signals to adhesion and virulence during bacterial infections. Moreover, the Dps-like proteins have lately attracted considerable interest in the field of nanotechnology owing to their ability to act as protein cages for iron and various other metals. A better understanding of their function and mechanism could therefore lead to novel applications in biotechnology and nanotechnology.     

RNA uridylyltransferases

The terminal RNA uridylyltransferases (TUTases) catalyze transfer of UMP residues to the 3' hydroxyl group of RNA. These activities are widespread among eukaryotes and appear to be involved in a variety of RNA-processing pathways. Recent studies of RNA editing in trypanosomatids have provided the first insights into the biological functions of RNA uridylyltransferases, which had eluded biochemical identification despite 30-year-old evidence of such activities in mammals and plants. Comparative sequence analysis of trypanosomal TUTases and their homologs revealed by large-scale genomic projects demonstrates a significant level of biochemical and structural diversity between putative uridylyltransferases. The conserved catalytic domain has acquired additional protein modules and appears to have adapted to perform functionally distinct tasks of guided U-insertion into mRNA and constrained addition of an oligo[U] tail to guide RNAs. Here I discuss the current knowledge of this novel enzyme family and possible roles of RNA uridylylation in the regulation of gene expression.     

Cell penetrating peptides: overview and applications to the delivery of oligonucleotides

Crossing biological barriers represents a major limitation for clinical applications of biomolecules such as nucleic acids, peptides or proteins. Cell penetrating peptides (CPP), also named protein transduction domains, comprise short and usually basic amino acids-rich peptides originating from proteins able to cross biological barriers, such as the viral Tat protein, or are rationally designed. They have emerged as a new class of non-viral vectors allowing the delivery of various biomolecules across biological barriers from low molecular weight drugs to nanosized particles. Encouraging data with CPP-conjugated oligonucleotides have been obtained both in vitro and in vivo in animal models of diseases such as Duchenne muscular dystrophy. Whether CPP-cargo conjugates enter cells by direct translocation across the plasma membrane or by endocytosis remains controversial. In many instances, however, endosomal escape appears as a major limitation of this new delivery strategy.     

Bioportide: an emergent concept of bioactive cell-penetrating peptides

Cell-penetrating peptides (CPPs) have proven utility for the highly efficient intracellular delivery of bioactive cargoes that include peptides, proteins, and oligonucleotides. The many strategies developed to utilize CPPs solely as pharmacokinetic modifiers necessarily requires them to be relatively inert. Moreover, it is feasible to combine one or multiple CPPs with bioactive cargoes either by direct chemical conjugation or, more rarely, as non-covalent complexes. In terms of the message-address hypothesis, this combination of cargo (message) linked to a CPP (address) as a tandem construct conforms to the sychnological organization. More recently, we have introduced the term bioportide to describe monomeric CPPs that are intrinsically bioactive. Herein, we describe the design and biochemical properties of two rhegnylogically organized monometic CPPs that collectively modulate a variety of biological and pathophysiological phenomena. Thus, camptide, a cell-penetrant sequence located within the first intracellular loop of a human calcitonin receptor, regulates cAMP-dependent processes to modulate insulin secretion and viral infectivity. Nosangiotide, a bioportide derived from endothelial nitric oxide synthase, potently inhibits many aspects of the endothelial cell morphology and movement and displays potent anti-angiogenic activity in vivo. We conclude that, due to their capacity to translocate and target intracellular signaling events, bioportides represent an innovative generic class of bioactive agents.     

Do natural DNA triple-helical structures occur and function in vivo?

Formation of natural intramolecular triple-helical structures of DNA is still an intriguing research topic in view of the possible involvement of these structures in biological processes. The biochemical and biophysical properties of DNA triplex structures have been extensively studied, and experimental data show that H-DNA is likely to form in vivo and may regulate the expression of various genes. However, direct and unambiguous evidence of the possible biological roles of these structures is yet elusive. This review focuses on the basic facts that are in favor of, or against, the hypothesis of the presence and function of natural DNA triple-helical structures in vivo, and outlines the different methods and probes that have been used to support these facts.     

Antisense-mediated redirection of mRNA splicing

Antisense technology has been used to study basic biological processes, and to block these processes when they deleteriously lead to human disease. A separate, equally important application of antisense technology is to upregulate the gene expression lost in the diseased state by shifting alternative splicing of pre-messenger RNA. This strategy has commonly relied upon the use of antisense oligonucleotides; however, another approach is to use a plasmid construct to generate antisense RNA inside the cell. Antisense therapeutics based on expression vectors and viral vectors offers a gene therapy approach, whereas those based on oligonucleotides offers a more drug like approach.     

Crystal structures of eukaryote glycosyltransferases reveal biologically relevant enzyme homooligomers

Glycosyltransferases (GTases) transfer sugar moieties to proteins, lipids or existing glycan or polysaccharide molecules. GTases form an important group of enzymes in the Golgi, where the synthesis and modification of glycoproteins and glycolipids take place. Golgi GTases are almost invariably type II integral membrane proteins, with the C-terminal globular catalytic domain residing in the Golgi lumen. The enzymes themselves are divided into 103 families based on their sequence homology. There is an abundance of published crystal structures of GTase catalytic domains deposited in the Protein Data Bank (PDB). All of these represent either of the two main characteristic structural folds, GT-A or GT-B, or present a variation thereof. Since GTases can function as homomeric or heteromeric complexes in vivo, we have summarized the structural features of the dimerization interfaces in crystal structures of GTases, as well as considered the biochemical data available for these enzymes. For this review, we have considered all 898 GTase crystal structures in the Protein Data Bank and highlight the dimer formation characteristics of various GTases based on 24 selected structures.     

Messenger RNA differential display strategies in birds and amphibians

As an inroad to the discovery of genes involved in important biological activities, various techniques have been developed for detecting genes based on their expression levels. Arbitrary amplification of different messenger RNA (mRNA) populations and their comparison on display autoradiograms made mRNA differential display one of the most straightforward approaches to identification of differentially regulated mRNAs. Over the past decade this strategy has been employed in many in vitro and in vivo systems. The use of the method in bird and amphibian model systems is reviewed here, emphasizing several unique combinations of model system and design of differential display screen.     

Biochemical aspects of radiation biology

In order to analyze the mechanisms of biological radiation effects, the events after radiation energy absorption in irradiated organisms have to be studied by physico-chemical and biochemical methods. The radiation effects in vitro on biomolecules, especially DNA, are described, as well as their alterations in irradiated cells. Whereas in vitro, in aqueous solution, predominantly OH radicals are effective and lead to damage in single moieties of the DNA, in vivo the direct absorption of radiation energy leads to 'locally multiply-damaged sites', which produce DNA double-strand breaks and locally denatured regions. DNA damage will be repaired in irradiated cells. Error free repair leads to the original nucleotide sequence in the genome by excision or by recombination. "Error prone repair"(mutagenic repair), leads to mutation. However, the biochemistry of these processes, regulated by a number of genes, is poorly understood. In addition, more complex reactions, such as gene amplification and transposition of mobile gene elements, are responsible for mutation or malignant transformation.     

AS-48 bacteriocin: close to perfection

Bacteriocin AS-48 is an intriguing molecule because of its unique structural characteristics, genetic regulation, broad activity spectrum, and potential biotechnological applications. It was the first reported circular bacteriocin and has been undoubtedly the best characterized for the last 25 years. Thus, AS-48 is the prototype of circular bacteriocins (class IV), for which the structure and genetic regulation have been elucidated. This review discusses the state-of-the-art in genetic engineering with regard to this circular protein, with the use of site-directed mutagenesis and circular permutation. Mutagenesis studies have been used to unravel the role of (a) different residues in the biological activity, underlining the relevance of several residues involved in membrane interaction and the low correlation between stability and activity and (b) three amino acids involved in maturation, providing information on the specificity of the leader peptidase and the circularization process itself. To investigate the role of circularity in the stability and biological properties of the enterocin AS-48, two different ways of linearization have been attempted: in vitro by limited proteolysis experiments and in vivo by circular permutation in the structural gene as-48A. The results summarized here show the significance of circularization on the secondary structure, potency and, especially, the stability of AS-48 and point as well to a putative role of the leader peptide as a protecting moiety in the pre-proprotein. Taken all together, the data available on circular bacteriocins support the idea that AS-48 has been engineered by nature to make a remarkably active and stable protein with a broad spectrum of activity.     

An overview of RNAs with regulatory functions in gram-positive bacteria

During the last decade, RNA molecules with regulatory functions on gene expression have benefited from a renewed interest. In bacteria, recent high throughput computational and experimental approaches have led to the discovery that 10–20% of all genes code for RNAs with critical regulatory roles in metabolic, physiological and pathogenic processes. The trans-acting RNAs comprise the noncoding RNAs, RNAs with a short open reading frame and antisense RNAs. Many of these RNAs act through binding to their target mRNAs while others modulate protein activity or target DNA. The cis-acting RNAs include regulatory regions of mRNAs that can respond to various signals. These RNAs often provide the missing link between sensing changing conditions in the environment and fine-tuning the subsequent biological responses. Information on their various functions and modes of action has been well documented for gram-negative bacteria. Here, we summarize the current knowledge of regulatory RNAs in gram-positive bacteria.