Algorithms for the search of amino acid patterns in nucleic acid sequences.

Some algorithms are described for the search of regions in a nucleic acid sequence that, when translated into amino acids, are homologous to a given amino acid pattern. All algorithms are modifications of the dynamic programming method for sequence comparison such that the translation of codons is taken into account. One of the algorithms has been implemented as a FORTRAN 77 program. The program operates on files that follow the format of the EMBL Nucleotide Sequence Data Library.     

Molecular Biocomputing Suite: a word processor add-in for the analysis and manipulation of nucleic acid and protein sequence data.

In all fields of molecular biology, researchers are increasingly challenged by experiments planned and evaluated on the basis of nucleic acid and protein sequence data generally retrieved from public databases. Despite the wide spectrum of available Web-based software tools for sequence analysis, the routine use of these tools has disadvantages, particularly because of the elaborate and heterogeneous ways of data input, output, and storage. Here we present a Visual Basic-encoded Microsoft Word Add-In, the Molecular BioComputing Suite (MBCS), available at the BioTechniques Software Library (www.BioTechniques.com). The MBCS software aims to manage and expedite a wide range of sequence analyses and manipulations using an integrated text editor environment including menu-guided commands. Its independence of sequence formats enables MBCS to be used as a pivotal application between other software tools for sequence analysis, manipulation, annotation, and editing.     

Interactive Fluorophore and Quencher Pairs for Labeling Fluorescent Nucleic Acid Hybridization Probes

The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.     

GENEUS, a computer system for DNA and protein sequence analysis containing an information retrieval system for the EMBL data library.

We describe a comprehensive computer system, GENEUS, for extensive DNA, RNA and protein sequence analysis. The analysis system is developed for the DEC VAX/VMS computer and uses the EMBL Nucleic Acid Sequence Data Library. Help information is available on-line on terminal screen. To speed up system handling, a qualifier oriented user communication is employed. All results are stored on files making them accessible to the computer editor. An information retrieval system for the EMBL Nucleotide Sequence Data Library is also described. A defined data-base interface allows connection to other analysis programs.+     

The extended left-handed helix: a simple nucleic acid-binding motif

The poly-proline type II extended left-handed helical structure is well represented in proteins. In an effort to determine the helix's role in nucleic acid recognition and binding, a survey of 258 nucleic acid-binding protein structures from the Protein Data Bank was conducted. Results indicate that left-handed helices are commonly found at the nucleic acid interfacial regions. Three examples are used to illustrate the utility of this structural element as a recognition motif. The third K homology domain of NOVA-2, the Epstein-Barr nuclear antigen-1, and the Drosophila paired protein homeodomain all contain left-handed helices involved in nucleic acid interactions. In each structure, these helices were previously unidentified as left-handed helices by secondary structure algorithms but, rather, were identified as either having small amounts of hydrogen bond patterns to the rest of the protein or as being "unstructured." Proposed mechanisms for nucleic acid interactions by the extended left-handed helix include both nonspecific and specific recognition. The observed interactions indicate that this secondary structure utilizes an increase in protein backbone exposure for nucleic acid recognition. Both main-chain and side-chain atoms are involved in specific and nonspecific hydrogen bonding to nucleobases or sugar-phosphates, respectively. Our results emphasize the need to classify the left-handed helix as a viable nucleic acid recognition and binding motif, similar to previously identified motifs such as the helix-turn-helix, zinc fingers, leucine zippers, and others.     

Nucleic acid hybridization: from research tool to routine diagnostic method

The nucleic acid hybridization reaction is extremely specific and thus a valuable tool for the identification of genes or organism of interest. The increasing use of nucleic acid hybridization in applied fields like diagnostic medicine has led to the development of more convenient hybridization assays than those originally used in basic research. In conventional nucleic acid hybridization methods immobilized nucleic acids are detected on a filter by a radiolabelled probe. Sandwich hybridization is a simple test format for the analysis of unpurified biological material, but has the disadvantage of a slow reaction rate. Solution hybridization methods are fast and easy to perform provided that a method to separate the formed hybrids from the reaction mixture is available. In non-isotopic detection the nucleic acid probe is modified with a chemical group, which is identified with a labelled detector molecule after hybridization. The low sensitivity of detection is the main problem in nucleic acid hybridization methods. Procedures to amplify the detectable signal or the amount of detectable nucleic acid sequences are potential solutions to this problem. The new hybridization methods have successfully been used for some applications, but still need to be combined into well performing tests to be applicable to any desired purpose.     

PAPQMD/AM1 Parametrization of the bonded term of aromatic biomolecules

The suitability of the PAPQMD/AM1 (Program for Approximate Parametrization from Quantum Mechanical Data/based on AM1 calculations) strategy to provide force-field parameters for large heteroaromatic compounds was studied. For this purpose, PAPQMD/AMI stretching and bending parameters for adenine, cytosine, thymine, guanine, and uracil were computed and compared with experimentally derived force-field parameters. Furthermore, equilibrium geometries and vibrational spectra for the five nucleic acid bases obtained from molecular mechanical calculations using both PAPQMD/AM1 and AMBER (Assisted Model Building with Energy Refinement) All Atoms force fields were compared with the available experimental data. © 1994 John Wiley & Sons, Inc.     

De novo design of sequences for nucleic acid structural engineering

An interactive procedure has been developed to assign sequences for the design of nucleic acid secondary structure. The primary goal of the procedure is to facilitate macromolecular architecture studies through the design of branched nucleic acid mono- and oligo-junction constructs in a convenient fashion. The essential feature of the sequence-symmetry minimization algorithm employed is the treatment of short sequences as vocabulary elements whose repetition decreases control over the resulting secondary structure. Both manual and semi-automatic application of this approach are available. The design of linear nucleic acid molecules or molecules containing single-stranded loops or connectors is also possible through application of the procedure.     

Molecular Probe Data Base (MPDB).

Molecular Probe Data Base contains detailed information on synthetic oligonucleotides with a sequence of up to 100 nucleotides. This database prevalently contains information related to human oligonucleotides used in diagnostics. Molecular Probe Data Base has been made available on-line through the Internet by means of Network Information Retrieval (NIR) tools since 1993. Two years ago, a collaboration with EMBL Data Library was also set up, so that the Molecular Probe Data Base has been integrated with other molecular biology data banks in the sphere of the SRS WWW network browser. In this paper, the most recent enhancements and the current status of the Molecular Probe Data Base are briefly presented.     

Biotin-streptavidin-labeled oligonucleotides as probes of helicase mechanisms

Helicases use the energy from ATP hydrolysis to catalyze formation of single-stranded nucleic acids by unwinding double-stranded nucleic acids. The ATP-dependent reaction can be broken down into at least two steps: melting of the duplex and translocation of the enzyme along the nucleic acid lattice. Each step presents difficulties for study because clear end points for the reactions are not always available. For example, translocation involves the movement of the enzyme from one point along the lattice to a new position, with no net change in chemical structure of the nucleic acid. Hence, new assays have been developed in which the nucleic acid is modified to contain a "protein block" that impedes translocation of the enzyme. To prepare such protein blocks, biotin-streptavidin has been used due to the ease with which the biotin can be incorporated into nucleic acids by chemical synthesis. Several applications of oligonucleotides labeled with biotin-streptavidin for the study of helicase mechanisms are described.     

Locked Nucleic Acid Pentamers as Universal PCR Primers for Genomic DNA Amplification

Background

Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can save a lot of time, cost and labor compared to traditional single reaction detection methods. However, the multiplexing method currently used requires precise handiwork and many complicated steps, making a new, simpler technique desirable. Oligonucleotides containing locked nucleic acid residues are an attractive tool because they have strong affinities for their complementary targets, they have been used to avoid dimer formation and mismatch hybridization and to enhance efficient priming. In this study, we aimed to investigate the use of locked nucleic acid pentamers for genomic DNA amplification and multiplex genotyping.

Results

We designed locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification. The locked nucleic acid pentamers were able to prime amplification of the selected sequences within the investigated genomes, and the resulting products were similar in length to those obtained by restriction digest. In Real Time PCR of genomic DNA from three bacterial species, locked nucleic acid pentamers showed high priming efficiencies. Data from bias tests demonstrated that locked nucleic acid pentamers have equal affinities for each of the six genes tested from the Klebsiella pneumoniae genome. Combined with suspension array genotyping, locked nucleic acid pentamer-based PCR amplification was able to identify a total of 15 strains, including 3 species of bacteria, by gene- and species-specific probes. Among the 32 species used in the assay, 28 species and 50 different genes were clearly identified using this method.

Conclusion

As a novel genomic DNA amplification, the use of locked nucleic acid pentamers as universal primer pairs in conjunction with suspension array genotyping, allows for the identification of multiple distinct genes or species with a single amplification procedure. This demonstrates that locked nucleic acid pentamer-based PCR can be utilized extensively in pathogen identification.     

E-MSD: the European Bioinformatics Institute Macromolecular Structure Database

The E-MSD macromolecular structure relational database (http://www.ebi.ac.uk/msd) is designed to be a single access point for protein and nucleic acid structures and related information. The database is derived from Protein Data Bank (PDB) entries. Relational database technologies are used in a comprehensive cleaning procedure to ensure data uniformity across the whole archive. The search database contains an extensive set of derived properties, goodness-of-fit indicators, and links to other EBI databases including InterPro, GO, and SWISS-PROT, together with links to SCOP, CATH, PFAM and PROSITE. A generic search interface is available, coupled with a fast secondary structure domain search tool.     

Nucleic acid visualization with UCSF Chimera

With the increase in the number of large, 3D, high-resolution nucleic acid structures, particularly of the 30S and 50S ribosomal subunits and the intact bacterial ribosome, advancements in the visualization of nucleic acid structural features are essential. Large molecular structures are complicated and detailed, and one goal of visualization software is to allow the user to simplify the display of some features and accent others. We describe an extension to the UCSF Chimera molecular visualization system for the purpose of displaying and highlighting nucleic acid characteristics, including a new representation of sugar pucker, several options for abstraction of base geometries that emphasize stacking and base pairing, and an adaptation of the ribbon backbone to accommodate the nucleic acid backbone. Molecules are displayed and manipulated interactively, allowing the user to change the representations as desired for small molecules, proteins and nucleic acids. This software is available as part of the UCSF Chimera molecular visualization system and thus is integrated with a suite of existing tools for molecular graphics.     

Clay-Nucleic Acid Complexes: Characteristics and Implications for the Preservation of Genetic Material in Primeval Habitats

The equilibrium adsorption of three nucleic acids: chromosomal DNA, supercoiled plasmid DNA, and 25S rRNA, on the clay minerals, montmorillonite (M) and kaolinite (K), were studied. Adsorption of the nucleic acid on the clays was rapid and maximal after 90 min of contact time. Chromosomal DNA was adsorbed to a greater extent than plasmid DNA and RNA, and the adsorption was also greater on M than on K. Adsorption isotherms were of the L type, and a plateau was reached with all the complexes, with the exception of chromosomal DNA adsorbed on M. To determine where nucleic acids are adsorbed on clay minerals and the nature of the interaction, complexes were studied by X-ray diffraction (X-RD), electron microscopy, and Fourier transform infrared (FT-IR) spectroscopy. X-RD showed that nucleic acids did not penetrate the clay, indicating that the adsorption occurred primarily on the external surfaces of clay particles, as also suggested by electron microscopy observations. FT-IR spectra of clay-tightly bound nucleic acid complexes showed absorption bands that indicate a variation of the nucleic acids status as a consequence of their adsorption on clay. Data obtained suggested that the formation of clay-nucleic acid complex could have an important role in the preservation of genetic material in primeval habitats.     

A procedure for simultaneous preparation of large amounts of DNA and RNA by the use of potassium iodide gradients.

A method is described that permits the isolation of both DNA and RNA from tissues on KI density gradients without destroying either nucleic acid species. Up to 3 mg can be resolved efficiently in a single 13-ml gradient. Data are presented which show that direct monitoring of the OD of nucleic acids in KI solutions is possible without addition of ethidium bromide. Furthermore, equations for the correlation of density, refractive index and weight per volume are presented.     

Protein structural models for nucleic acid interactions

It is proposed that particular segments of some ribosomal, histone and plant viral capsid proteins adopt a helical structural mode for interaction with nucleic acid. The amino acid regions were determined by three probes applied to 26 protein sequences: searches for helical wheels displaying asymmetric basic charge distributions, secondary structural predictions, and searches for primary structural homologies. In 11 of the protein sequences examined, homologous heptapeptides were found in the residue spans delineated by the three probes. A helical wheel analysis of the oligopeptide amino acids showed a distinct positive charge clustering. It is suggested that the basic amino acid side chains on the hydrophilic helical side interact with nucleic acid negative phosphate groups while the somewhat hydrophobic side is available for interaction within the protein or possibly with the major groove of double-stranded nucleic acid.     

Nucleic acid carriers based on precise polymer conjugates

Polymer polydispersity, random conjugation of functional groups, and poorly understood structure-activity relationships have constantly hampered progress in the development of nucleic acid carriers. This review focuses on the synthetic concepts for the generation of precise polymers, site-specific conjugation strategies, and multifunctional conjugates for nucleic acid transport. Dendrimers, defined peptide carriers, sequence-defined polyamidoamines assembled by solid-phase supported synthesis, and precise lipopeptides or lipopolymers have been characterized for pDNA and siRNA delivery. Conjugation techniques such as click chemistries and peptide ligation are available for conjugating polymers with functional transport elements such as targeting or shielding domains and for direct covalent modification of therapeutic nucleic acids in a site-specific mode.     

DNAssist: the integrated editing and analysis of molecular biology sequences in windows

MOTIVATION: The programs currently available for the analysis of nucleic acid and protein sequences suffer from a variety of problems: Web-based programs often require inconvenient reformatting of sequences when proceeding from one analysis to the next, and commercial-console-based programs are cost prohibitive. Here, we report the development of DNASSIST:, an inexpensive, multiple-document, interface program for the fully integrated editing and analysis of nucleic acid and protein sequences in the familiar environment of Microsoft Windows.