A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA.

A method is presented for choosing optimal oligodeoxyribonucleotides as probes for filter hybridization, primers for sequencing, or primers for DNA amplification. Three main factors that determine the quality of a probe are considered: stability of the duplex formed between the probe and target nucleic acid, specificity of the probe for the intended target sequence, and self-complementarity. DNA duplex stability calculations are based on the nearest-neighbor thermodynamic values determined by Breslauer et al. [Proc. Natl. Acad. Sci. U.S.A. (1986), 83: 3746]. Temperatures of duplex dissociation predicted by the method described here were within 0.4 degrees C of the values obtained experimentally for ten oligonucleotides. Calculations for specificity of the probe and its self-complementarity are based on a simple dynamic algorithm.     

Unusually stable β-sheet formation in an ionic self-complementary oligopeptide

A 16-residue amphiphilic oligopeptide (EAK16) with every other residue alanine and also containing glutamic acid and lysine (Ac-NH-AEAEAKAKAEAEAKAK-CONH₂) is able to form an unusually stable β-sheet structure. The β-sheet structure is stable at very low concentrations in water and at high temperatures. Various pH changes at 1.5, 3, 7, and 11 had little effect on the stability of the β-sheet structure. The β-sheet structure was not altered significantly even in the presence of 0.1% SDS, 7 molar guanidine hydrochloride, or 8 molar urea. One of the structural characteristics of the EAK16 is its ionic self-complementarity in that ionic bonds and hydrogen bonds between Glu and Lys can form readily between two oligopeptide β-sheet structures. This structural feature is probably one of the factors that promotes its extreme stability. This is the first example of such an extended ionic self-complementarity in a protein structure. EAK16 and its related peptides may have applications as useful biomaterials. It also offers a good model for studying the mechanism of β-sheet formation. Because the oligopeptide can self-assemble to form a membranous structure, it may have relevance to origin of life research. © 1994 John Wiley & Sons, Inc.     

Properties of the histidines of chymotrypsinogen: comparison with alpha-chymotrypsin

Adeno-associated virus linear, single polynucleotide chains contain an inverted terminal repetition which allows the formation of single-stranded circles when the DNA is exposed to annealing conditions. Under appropriate annealing conditions single-stranded circular dimers are formed, the majority of which have two projections separated by 180 ° visible in the electron microscope. We conclude that these projections represent the regions of self-complementarity (inverted terminal repetition) contained within the virus DNA. Measurements of the lengths of the projections indicate that the length of the inverted terminal repetition represents approximately 1.5% of the genome.     

Informational analysis of MS2 and θX174 virus genomes

In this communication we compare the amount of independent and dependent infonnation of two different structures of virus genomes: that of MS₂, able to display high secondary structure, and that of θX174, with scarce self-complementarity. The references for this comparison were the average value of informational indexes and the ability to generate secondary structure of the well known transfer tRNAs. The analysis of these parameters reveals the singular behaviour of each species, which obtains a high reliable genetic information by different molecular arrangements.     

applications of electroporation of adherent cellsIn Situ,on a partly conductive slide

One of the most important factors affecting the quality of PCR is the choice of primers. In general, the longer the PCR product the more difficult it is to select efficient primers and set appropriate designing primers, and in general, the more DNA sequence information is available, the better the ch0ance of finding an optimal primer pair. Efficient primers can be designed by avoiding the following flaws: primer-dimer formation, self-complementarity, too lowT _m of the primers, and/or their incorrect internal stability profile. Tips on subcloning PCR products, calculating duplex stability (predicting dimer formation strength), and designing degenerate primers are given.     

Calnexin Delta 185-520 partially reverses the misprocessing of the Delta F508 cystic fibrosis transmembrane conductance regulator

We analyzed the interrelation between the efficiency of a gene expression and the nucleotide composition of all protein-coding sequences in 38 unicellular organisms whose complete genomic sequences are known. These organisms comprise 37 prokaryotic (29 eubacteria and eight archaebacteria) and one eukaryotic (yeast) species. We demonstrated that frequency analysis of gene codon composition fails to reflect adequately the gene expression efficiency of all these organisms. We constructed a measure, the elongation efficiency index, that considers simultaneously the information on codon frequencies and the degree of mRNA local self-complementarity. This measure recognizes the ribosome-coding genes as highly expressed in all the unicellular organisms studied. According to our analysis, these species fall into five groups differentiated by the process that makes the key contribution to the elongation rate.     

Activation of Autoreactive B Cells by Endogenous TLR7 and TLR3 RNA Ligands

The key step in the activation of autoreactive B cells is the internalization of nucleic acid containing ligands and delivery of these ligands to the Toll-like Receptor (TLR) containing endolysosomal compartment. Ribonucleoproteins represent a large fraction of autoantigens in systemic autoimmune diseases. Here we demonstrate that many uridine-rich mammalian RNA sequences associated with common autoantigens effectively activate autoreactive B cells. Priming with type I IFN increased the magnitude of activation, and the range of which RNAs were stimulatory. A subset of RNAs that contain a high degree of self-complementarity also activated B cells through TLR3. For the RNA sequences that activated predominantly through TLR7, the activation is proportional to uridine-content, and more precisely defined by the frequency of specific uridine-containing motifs. These results identify parameters that define specific mammalian RNAs as ligands for TLRs.     

The nucleotide sequences at the termini of adenovirus-2 DNA.

The nucleotide sequence of the first 156 residues from the left end and the first 134 residues from the right end of adenovirus-2 DNA have been determined by direct DNA sequencing techniques. The inverted terminal repetition is 102 nucleotide pairs long. The 5′-ends of the intact DNA are resistant to the action of T4 polynucleotide kinase and the 5′ → 3′ exonucleases from phages lambda and T7. This resistance is most likely due to the covalent attachment of the 5′-terminal C residue to the terminal protein. No significant self-complementarity exists within the inverted terminal repetition, making terminal initiation of DNA replication via a self-priming mechanism unlikely. However, the terminal A + T-rich region followed immediately by a very G + C-rich region is consistent with other schemes for adenovirus-2 DNA replication. The left end of adenovirus-2 DNA contains extensive sequence repetition.     

Molecular basis of action of HyBeacon fluorogenic probes: a spectroscopic and molecular dynamics study

HyBeacons, novel DNA probes for ultra-rapid detection of single nucleotide polymorphisms, contain a fluorophore covalently attached via a linker group to an internal nucleotide. As the probe does not require a quencher or self-complementarity to function, this study investigates the molecular-level mechanism underlying the increase of fluorescence intensity on hybridization of HyBeacons with target DNA. Spectroscopic ultraviolet-visible and fluorimetric studies, combined with molecular dynamics simulations, indicate projection of the fluorophore moiety away from the target-probe duplex into aqueous solution, although specific linker-DNA interactions are populated. Based on evidence from this study, we propose that for HyBeacons, the mechanism of increased fluorescence on hybridization is due to disruption of quenching interactions in the single-stranded probe DNA between the fluorophore and nucleobases. Hybridization leads to an extended linker conformation, removing the fluorophore from the immediate vicinity of the DNA bases.     

Bioanalytical characterization of proteins

Allergens from the view of a protein chemist are quite normal proteins, not to distinguish from non allergenic proteins. The first task is therefore to recognize and identify the proteins responsible for the allergenic reaction. This is usually only possible if the allergenic structure is conserved during the purification procedures. For a detailed analysis of the allergenic protein modern protein chemical methods for characterization, identification, determination of posttranslational modifications and epitope characterization have to be applied. Such techniques are briefly described in this article.     

Characterization of the bovine prothrombin gene

The bovine prothrombin gene was characterized by Southern blot analysis of bovine genomic DNA using bovine prothrombin cDNA fragments as hybridization probes. These analyses suggested that the bovine genome contains a single prothrombin gene that is at least 10 kilobase pairs (kbp) in size. To characterize the gene more thoroughly, two bovine genomic phage libraries were screened by using prothrombin cDNAs as hybridization probes. Heteroduplex analysis of the cloned genomic DNA and cDNA showed that the prothrombin gene is 14.9 kbp in size and contains at least 14 exons interrupted by 13 introns. The exons vary in size from 28 to 317 base pairs (bp), while the introns vary in size from less than 100 to 6940 bp. Regions of self-complementarity were observed within some of the introns, suggesting the presence of inverted repeat sequences. The bovine prothrombin gene shows similarities in structure to both the human prothrombin gene and the human factor IX gene.     

BIGPROBE: a computer program that predicts the sequence of long oligonucleotide probes with high reliability

We have written a computer program, BIGPROBE, which facilitates the design of long nucleic acid probes from the partial or complete amino acid sequence of a protein. BIGPROBE relies upon information on codon usage, intercodon dinucleotide frequency, and potential probe self-complementarity. We have examined the accuracy with which the program predicts coding sequences using sample human and rat genes and probe lengths of 30-60 nucleotides. Rat probe sequences selected by BIGPROBE using either codon usage or dinucleotide frequency data alone averaged 86-92% homology with the known exons of the corresponding gene sequences. Predictive accuracy with rat gene probes could be improved to 89-94%, depending upon probe length, by applying codon usage and dinucleotide frequency data in combination. Similar accuracy was achieved for human genes.     

Salt-independent adsorption of human serum proteins on cyanocarbon gels

Electron donor acceptor gels based on cyanocarbons have been tested for human serum protein adsorption in the absence of salt-promotion by water-structuring salt. This phenomenon was compared with a normal adsorption process in the presence of salt. The tricyanoaminopropene–divinyl sulfone–agarose displayed unusual protein adsorption properties as binding could occur both independently or dependently of the salt-promotion. The absence of hydrophobic or ionic character of the salt-independent interaction suggests an electron donor acceptor adsorption mechanism which is shown, for the first time, to occur independently of salt-promotion in aqueous solution. Study of the protein adsorption specificity showed similar protein selectivity for the fractions adsorbed in both conditions.     

Three-step purification of lipopolysaccharide-free, polyhistidine-tagged recombinant antigens of Mycobacterium tuberculosis

Previous work has shown that the study of host immune responses against Mycobacterium tuberculosis, the causative agent of tuberculosis, requires the availability of multiple mycobacterial antigens. Since purification of protein from M. tuberculosis cells is extremely cumbersome, we developed a protocol for purifying milligram amounts of ten recombinant antigens of M. tuberculosis from E. coli cells. Purified proteins were immunologically active and free of contaminants that confound interpretation of cell-based immunological assays. The method utilizes a three-step purification protocol consisting of immobilized metal-chelate affinity chromatography, size exclusion chromatography and anion-exchange chromatography. The first two chromatographic steps yielded recombinant protein free of protein contaminants, while the third step (anion-exchange chromatography) efficiently removed E. coli lipopolysaccharide, a potent polyclonal activator of lymphoid cells. The recombinant proteins were immunologically indistinguishable from their native (i.e., purified from M. tuberculosis) counterparts. Thus the method provides a way to utilize recombinant proteins for immunological analyses that require highly purified antigens.     

Fast large scale oligonucleotide selection using the longest common factor approach

We present a fast method that selects oligonucleotide probes (such as DNA 25-mers) for microarray experiments on a truly large scale. For example, reliable oligos for human genes can be found within four days, a speedup of one to two orders of magnitude compared to previous approaches. This speed is attained by using the longest common substring as a specificity measure for candidate oligos. We present a space- and time-efficient algorithm, based on a suffix array with additional information, to compute matching statistics (lengths of longest matches) between all candidate oligos and all remaining sequences. With the matching statistics available, we show how to incorporate constraints such as oligo length, melting temperature, and self-complementarity into the selection process at a postprocessing stage. As a result, we can now design custom oligos for any sequenced genome, just as the technology for on-site chip synthesis is becoming increasingly mature.     

A NMR strategy to unambiguously distinguish nucleic acid hairpin and duplex conformations applied to a Xist RNA A-repeat

All RNA sequences that fold into hairpins possess the intrinsic potential to form intermolecular duplexes because of their high self-complementarity. The thermodynamically more stable duplex conformation is favored under high salt conditions and at high RNA concentrations, posing a challenging problem for structural studies of small RNA hairpin conformations. We developed and applied a novel approach to unambiguously distinguish RNA hairpin and duplex conformations for the structural analysis of a Xist RNA A-repeat. Using a combination of a quantitative HNN-COSY experiment and an optimized double isotope-filtered NOESY experiment we could define the conformation of the 26-mer A-repeat RNA. In contrast to a previous secondary structure prediction of a double hairpin structure, the NMR data show that only the first predicted hairpin is formed, while the second predicted hairpin mediates dimerization of the A-repeat by duplex formation with a second A-repeat. The strategy employed here will be generally applicable to identify and quantify populations of hairpin and duplex conformations and to define RNA folding topology from inter- and intra-molecular base-pairing patterns.