Molecular structure of nucleic acids. Molecular structure of deoxypentose nucleic acids. 1953.
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DNA↗
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Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid.
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DNA↗
Temperature-gradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions.
Temperature-gradient gel electrophoresis (TGGE) is applied to analyze conformational transitions and sequence variations of nucleic acids and protein-nucleic acid interactions. A linear and highly reproducible temperature-gradient is established perpendicular or parallel to the direction of the electrophoresis. The instrument consists of an electrically insulated metal plate, which is heated at one edge and cooled at the other edge by two thermostating baths and is used as an ancillary device for commercial horizontal gel electrophoresis instruments. Biopolymers are separated in TGGE according to size, shape and thermal stability of their conformational transitions. If the temperature-gradient is established perpendicular to the electrophoresis, monomolecular conformational transitions of nucleic acids show up as continuous transition curves; strand-separation leads to discontinuous transitions. In the studies on viroid RNA it was shown that natural circular viroid RNA undergoes one highly cooperative transition detected by TGGE as a drastic retardation in mobility. Oligomeric replication intermediates of viroids exhibit coexisting structures which could not be detected by any other technique. Double-stranded satellite RNA from cucumber mosaic virus is a mixture of sequence variants, all of which have the identical length of 335 nucleotides. In TGGE six different strains were resolved. Sequence variants of viroids were analyzed by hybridizing viroid RNA to (-)strand viroid RNA transcripts from viroid cDNA clones. Sequence variations lead to mismatches in the double strands and thereby to a shift of the transition curve to lower temperature. Mutations in plasmids, particularly in cloned inserts, were detected by mixing plasmids of two different clones, linearizing, denaturing, renaturing, and searching for shifts in the transition curves, which are generated by mismatch-formation during the renaturation of (+)- and (-)strands from different clones. Examples are given for different viroid clones and HIV-clones from one and the same patient. In another example, clones with point mutations from site-directed mutagenesis are analyzed and selected by TGGE. TGGE is also applied to study the effect of amino acid exchanges in the Tet repressor from E. coli on the thermal stability of the repressor and on the mode of binding of the repressor to the operator DNA. The results are discussed under the aspect that TGGE may be applied as routine analytical laboratory procedure.
Base Sequence↗
Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. J.D. Watson and F.H.C. Crick. Published in Nature, number 4356 April 25, 1953.
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DNA↗
Chemistry and structure of nucleic acids of bacteriophages. Many forms of nucleic acids of bacteriophages show the ways that information is stored and reproduced.
The nucleic acids of bacteriophages are characterized by a surprising multiformity. RNA and DNA may occur, the latter in single- or double-stranded form, circular or linear, with or without breaks or single-strand ends. Terminal redundancy may exist and the populations of linear phages may be uniform or randomly permuted. A double-stranded circular DNA does not occur in extracellular bacteriophage, but is often if not always formed after infection of the bacterial host. Phage DNA may be glucosylated or methylated to a certain extent, and the glucose and methyl residues may influence the stability of the DNA inside the host.
Bacteriophages↗
[X-ray structural studies of nucleic acid molecular structure and nucleic acid-protein interaction mechanism].
Since the discovery of the right-handed double helical structure of deoxyribonucleic acid by Watson and Crick in 1953, many pieces of detailed nucleic acid structural information involving ribonucleic acid have been elucidated and this review describes the results of the X-ray structural studies of nucleic acids and their constituents carried out in my laboratory. In the latter half the nucleic acid-protein interaction mechanism was also discussed on the basis of the molecular model of the stacking interaction between base and aromatic amino acid side chains. The structure-function relationship of ribonuclease T1.guanosine monophosphate complex was used as an example.
Binding Sites↗
Adaptive recognition by nucleic acid aptamers.
Nucleic acid molecules play crucial roles in diverse biological processes including the storage, transport, processing, and expression of the genetic information. Nucleic acid aptamers are selected in vitro from libraries containing random sequences of up to a few hundred nucleotides. Selection is based on the ability to bind ligand molecules with high affinity and specificity. Three-dimensional structures have been determined at high resolution for a number of aptamers in complex with their cognate ligands. Structures of aptamer complexes reveal the key molecular interactions conferring specificity to the aptamer-ligand association, including the precise stacking of flat moieties, specific hydrogen bonding, and molecular shape complementarity. These basic principles of discriminatory molecular interactions in aptamer complexes parallel recognition events central to many cellular processes involving nucleic acids.
Adenosine Monophosphate↗
Interaction of nucleic acids. VI. Interaction of purine with nucleic acids.
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Chemical Phenomena↗
Development of gene therapies for cardiovascular and renal diseases by nucleic acid medicines.
Nucleic acid medicines such as antisense DNA, antisense peptide nucleic acid (PNA), ribozyme, and decoy are expected to be novel therapeutic strategy for sever diseases which are resistant to present therapy. We have developed antisense DNA, antisense PNA and ribozyme targeting platelet-derived growth factor (PDGF) A-chain and transforming growth factor-beta1 (TGF-beta1) for arterial proliferative diseases such as coronary artery stenosis after angioplasty or stent implantation, hypertensive vascular diseases and atherosclerosis, and progressive renal diseases. Antisense DNA to PDGF A-chain inhibited arterial growth in spontaneously hypertensive rats without lowering blood pressure and inhibited the neointima formation of pig coronary artery after stent implantation. Ribozymes to PDGF A-chain and TGF-beta1 specifically inhibited the target transcripts and prevented the neointima formation. Ribozymes to TGF-beta1 improved renal damages in hypertensive rats. These nucleic acid medicines targeting PDGF A-chain and TGF-beta1 will be feasible gene therapies for the arterial proliferative diseases and progressive renal diseases. Pyrrole-imidazole polyamides are novel gene silencing compound, which bind to minor grove of double strand DNA by base-specific manner to inhibit gene expression. We developed pyrrole-imidazole polyamide to TGF-beta1 and confirmed that the polyamide binds to the TGF-beta1 promoter. The polyamide inhibited TGF-beta1 promoter activity and decreased expression of TGF-beta1 in vitro and in vivo. The polyamide markedly improved the renal injury in hypertensive rats. The pyrrole-imidazole polyamide will be a novel gene silencing agent for cardiovascular and renal diseases.
Amides↗
Optical absorption assay for strand-exchange reactions in unlabeled nucleic acids.
The nucleic acid exchange reaction is a common feature for genetic recombination, DNA replication and transcription. Due to the fact that in the strand-exchange reactions the reactant and product molecules have similar or identical nucleotide sequences, the reaction is undetectable. As a rule, the nucleic acids with radioactive or fluorescence labels are used in such studies. Besides the fact that the labels can perturb the reaction and pose a health risk to the investigators, the assays usually involve extra experimental steps: quenching the reaction, separation, visualization and quantification of the products. Here, we describe a straightforward, direct and precise method to study strand-exchange reaction of unlabeled nucleic acids by real-time measurements of optical absorption. The method takes advantage of the property of some guanine-rich oligonucleotides to adopt monomolecular quadruplex conformation in the presence of certain cations. The conformation is characterized by significant absorption in long-wavelength range of the ultraviolet region where usually other secondary structures are transparent. The 'signal' oligonucleotide is incorporated into reactant duplex by annealing with target sequence. Adding the replacement sequence initiates the release of the 'signal' oligonucleotide into solution, which is accompanied by ultraviolet absorption in long-wavelength range.
Aptamers, Nucleotide↗
Changes in immunodiffusion patterns and in nucleic acid content of Staphylococcus aureus grown in the presence of a nucleic acid fluorochrome.
Sonea, S. (University of Montreal, Montreal, Canada), J. de Repentigny, and A. Frappier. Changes in immunodiffusion patterns and in nucleic acid content of Staphylococcus aureus grown in the presence of a nucleic acid fluorochrome. J. Bacteriol. 84:1056-1060. 1962.-When grown in the presence of acridine orange, coagulase-positive Staphylococcus aureus exhibits changes in the number and the shape of its precipitin bands; these are more pronounced with commercial horse staphylococcal antitoxin than with normal human gamma-globulin. Simultaneously, there is an increase in the content of nucleic acids, especially deoxyribonucleic acid. Consequently, when grown in the presence of this nucleic acid fluorochrome, pathogenic staphylococci become similar to nonpathogenic strains in their antigenic structure and nucleic acid content. The bacterial population becomes a mixture of red and green fluorescing cells of different sizes. After centrifugation, most of the large and red fluorescing cells are found in the top layer, which shows more changes in antigenic composition and in nucleic acid content than the bottom layer. In subcultures grown in the absence of acridine orange, the cells revert to the original composition of the pathogenic strains.
Acridines↗
Triggers for switching from minipool testing by nucleic acid technology to individual-donation nucleic acid testing for West Nile virus: analysis of 2003 data to inform 2004 decision making.
BACKGROUND: Concern about West Nile virus (WNV) transfusion-transmitted infections missed by minipool (MP) nucleic acid testing (NAT) has prompted consideration of the use of individual-donation (ID) NAT. Strategies were investigated for the application of limited ID-NAT capacity in 2004. STUDY DESIGN AND METHODS: Patterns of WNV MP-NAT-reactive donations tested by the Blood Systems Laboratory each week for 79 blood centers from June 29 to November 23, 2003 (196 MP-NAT repeat-reactive [RR] donations among 801,697 units), were analyzed. ID-NAT initiation strategies were developed consisting of counts of RR donations and/or weekly RR rates, together with three ID-NAT discontinuation strategies, and ID testing burden was assessed based on these combined start and stop strategies. RESULTS: The effectiveness, reported as the percentage of MP-RR donations that would trigger ID-NAT based on each initiation strategy, ranged from 57 to 100 percent. The addition of a 1- or 2-week no-yield requirement for ID discontinuation substantially increased testing burden. Combined strategies resulted in projected ID-NAT of between 10 and 50 percent of donations for a 10- to 20-week period. For this organization, the most feasible ID-NAT initiation strategy was 2 MP-reactive donations and a weekly rate of 1 in 1000, which had an effectiveness of 81 percent and led to peak weekly ID-NAT of 20 to 25 percent of donations depending on the discontinuation rule. CONCLUSION: This new approach of targeted ID-NAT based on ongoing monitoring of MP-NAT yield may prove to be a rational policy for agents like WNV that cause seasonal and regional epidemics.
Blood Banks↗
Rapid, automated nucleic acid probe assays using silicon microstructures for nucleic acid concentration.
A system for rapid point-of-use nucleic acid (NA) analysis based on PCR techniques is described. The extraction and concentration of DNA from test samples has been accomplished utilizing silicon fluidic microchips with high surface-area-to-volume ratios. Short (500 bp) and medium size (48,000 bp) DNA have been captured, washed, and eluted using the silicon dioxide surfaces of these chips. Chaotropic (GuHCl) salt solutions were used as binding agents. Wash and elution agents consisted of ethanol-based solutions and water, respectively. DNA quantities approaching 40 ng/cm2 of binding area were captured from input solutions in the 100-1000 ng/mL concentration range. For dilute samples of interest for pathogen detection, PCR and gel electrophoresis were used to demonstrate extraction efficiencies of about 50 percent, and concentration factors of about 10x using bacteriophage lambda DNA as the target. Rapid, multichannel PCR thermal cycling modules with integrated solid-state detection components have also been demonstrated. These results confirm the viability of utilizing these components as elements of a compact, disposable cartridge system for the detection of NA in applications such as clinical diagnostics, biowarfare agent detection, food quality control, and environmental monitoring.
Automation↗
Linkage of pH, anion and cation effects in protein-nucleic acid equilibria. Escherichia coli SSB protein-single stranded nucleic acid interactions.
We have examined the linkage between pH and monovalent salt concentration (NaCl and NaF) on the equilibrium binding of the Escherichia coli SSB protein to single stranded poly(U) in its (SSB)65 binding mode. In this mode, single-stranded nucleic acid interacts with all four subunits of the SSB tetramer covering approximately 65 nucleotides and nearest-neighbor cooperative interactions can form between DNA bound SSB tetramers, although protein clusters are limited to dimers of tetramers (octamers). The intrinsic association equilibrium constant, K(obs), and the "limited" cooperativity parameter, omega T/O, have been determined from titrations that monitor the quenching of the SSB tryptophan fluorescence upon binding poly(U). The cooperativity parameter, omega T/O, is independent of salt concentration and type and increases only slightly with increasing pH. However, K(obs) decreases with increasing salt concentration due to a net release of ions accompanying complex formation. This net ion release has contributions from cation release from the nucleic acid as well as differential binding of both cations and anions to the protein. The dependence of K(obs) on [NaF] is independent of pH with delta logK(obs)/delta log[NaF] = -4.5(+/- 0.5). However, there is a strong linkage between the effects of [NaCl] and pH, such that (delta logK(obs)/delta log[NaCl]) ranges from -12.0(+/- 0.8) at pH 5.5, to -6.0(+/- 0.5) at pH 9.0 (at 25 degrees C). Thus Cl- release increases with decreasing pH due to a linkage between chloride binding and protonation of the protein, whereas there is essentially no release of F-. The linkages of ion concentration and pH on K(obs) can be described in terms of: (1) cation release from the polynucleotide; (2) release of Cl- from sites on the SSB tetramer that require protonation to bind Cl-; (3) binding of cations to sites on the SSB tetramer which require deprotonation for cation binding, and (4) required binding of two-to-three protons by the SSB tetramer in order to form the SSB-poly(U) complex. Thus, the influence of salt concentration on protein-nucleic acid equilibria can be quite complex with contributions from differential ion binding to both the protein and the nucleic acid; however, these can be resolved by examining the linked effects of pH and salt concentration on these interactions.
Anions↗
Investigation of the effect of cellular and viral nucleic acids on certain virus infections. Note 2. Effect of nucleic acids on virus multiplication in cell cultures.
Treatment of cell cultures with different natural nucleic acids prior to inoculation of herpes simplex virus type 1 led in certain cases to an obvious reduction in infectant titer. The reduction was maximum at a dose of 50 micrograms nucleic acid/culture tube and it was not dependent on the nature of the nucleic acid preparation. The antiviral effect of nucleic acids was enhanced by complexation with intercalation agents such as ethidium bromide or violamycin BI. No detectable amounts of interferon could be made evident in cell cultures treated with chromosomal DNA under conditions leading to a reduction by 1.75 log in infectant titer.
Aminoglycosides↗
Analysis of conformational parameters in nucleic acid fragments. II. Co-crystal complexes of nucleic acid bases.
Studies have been made of conformational parameters in co-crystal complexes and compounds of nucleic acid bases in which there is the possibility of formation of hetero-base-pairs. Using published data extracted from the Cambridge structural database, a total of 37 base-pairs were found, of which 25 were hetero-pairs and 12 homo-pairs. These base-pairs were subject to analysis to reveal hydrogen bond parameters, propeller twist, buckle and C1'-C1' separation (or a similar parameter if C1' atoms were not present). Hetero-pairs were found to show larger twists than homo-pairs, the magnitude of twist being unrelated to hydrogen bond parameters or buckle value. The propeller twisting is less pronounced in these nucleic acid bases than in nucleosides, but still has a significant magnitude. Propeller twisting in hetero-pairs is found to be larger than in homo-pairs. Hetero-pairs appear to be formed preferentially in competitive situations.
Crystallization↗
An introduction to peptide nucleic acid.
Peptide Nucleic Acid (PNA) is a powerful new biomolecular tool with a wide range of important applications. PNA mimics the behaviour of DNA and binds complementary nucleic acid strands. The unique chemical, physical and biological properties of PNA have been exploited to produce powerful biomolecular tools, antisense and antigene agents, molecular probes and biosensors.
DNA↗
Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification.
Nucleic acid amplification techniques are used for signal generation in antibody-based immunoassays, thereby dramatically enhancing the sensitivity of conventional immunoassays. Methodological aspects, as well as applications of this novel approach, are summarized in this review, with an emphasis on immuno-polymerase chain reaction (IPCR). IPCR is based on chimeric conjugates of specific antibodies and nucleic acid molecules, the latter of which are used as markers to be amplified by PCR for signal generation. The enormous efficiency of nucleic acid amplification typically leads to a 100-10,000-fold increase in sensitivity, as compared with the analogous enzyme-amplified immunoassay. The evolution of IPCR included the development of efficient reagents, the design of assay formats and the maintenance of functionality, even within complex biological matrices. Eventually, IPCR crossed the border from being a research method to a routine laboratory technique, enabling a broad range of applications in immunological research and clinical diagnostics.
Immunosorbent Techniques↗