RESUMEN
Oligonudeotide scanning arrays are useful tools in the study of nucleic acid interaction. Such arrays of oligonucleotides, corresponding to a full set of complements of a known sequence, can be readily made in a single series of coupling reactions, adding each nudeotide in the complement of the target sequence in turn. The synthesis is carried out on the surface of a solid substrate such as glass or polypropylene that has been modified to allow nucleotide coupling. A mask is used to apply the DNA synthesis reagents in a defined area and is moved by a fixed step size after each coupling reaction so that consecutive couplings overlap a portion of the previous one. The size of the mask and the displacement at each coupling determine the length of the oligonucleotides. A radiolabeled or fluorescently tagged target sequence is hybridised to a scanning array and its interaction with the complementary oligonudeotides is displayed as a hybridisation signal. It is thus possible to determine the exact sequence and lengths of large numbers of interacting sequences in a single hybridisation experiment. The array image is analysed using a computer program (xvseq) that calculates quantitative measurements of the binding strengths. We have found scanning arrays a useful tool not only to find effective antisense reagents, but also to study RNA folding and the mechanisms of heteroduplex formation. In this article, we discuss the format of these arrays, the technology used to fabricate and to read them, and their applications.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Oligonucleótidos Antisentido/química , ARN Catalítico/química , ARN/química , Tionucleótidos/química , Algoritmos , Diseño de Equipo , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/genética , Oligonucleótidos Antisentido/genética , ARN/genética , ARN Catalítico/genética , Análisis de Secuencia/instrumentación , Análisis de Secuencia/métodos , Tionucleótidos/genéticaRESUMEN
Transcripts representing mRNAs of three Xenopus cyclins, B1, B4 and B5, were hybridised to arrays of oligonucleotides scanning the first 120 nt of the coding region to assess the ability of the immobilised oligonucleotides to form heteroduplexes with their targets. Oligonucleotides that produced high heteroduplex yield and others that showed little annealing were assayed for their effect on translation of endogenous cyclin mRNAs in Xenopus egg extracts and their ability to promote cleavage of cyclin mRNAs in oocytes by RNase H. Excellent correlation was found between antisense potency and affinity of oligonucleotides for the cyclin transcripts as measured by the array, despite the complexity of the cellular environment.
Asunto(s)
Emparejamiento Base , Ciclinas/genética , Oligodesoxirribonucleótidos Antisentido/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , Xenopus laevis/genética , Animales , Sitios de Unión , Extractos Celulares , Clonación Molecular , Ciclinas/biosíntesis , Ciclinas/inmunología , Regulación de la Expresión Génica , Sueros Inmunes/inmunología , Cinética , Modelos Moleculares , Ensayos de Protección de Nucleasas , Hibridación de Ácido Nucleico , Oligodesoxirribonucleótidos Antisentido/química , Oligodesoxirribonucleótidos Antisentido/metabolismo , Oocitos/citología , Oocitos/metabolismo , Pruebas de Precipitina , ARN Mensajero/biosíntesis , ARN Mensajero/química , ARN Mensajero/metabolismo , Ribonucleasa H/metabolismo , Especificidad por SustratoRESUMEN
Selection of the appropriate target site is crucial to the success of an antisense experiment. The selection is difficult because RNAs fold to form secondary structures, rendering most of the molecule inaccessible to intermolecular base pairing with complementary nucleic acids. Conventional approaches, such as selection by 'sequence-walking' or computer-assisted design, have not brought significant success. Several empirical selection methods have been reported, a number of which are summarised in this review. Of notable significance are the 'global' methods based on mapping of transcripts with the endoribonuclease H (RNase H) and oligonucleotide scanning arrays.
Asunto(s)
Oligonucleótidos Antisentido/administración & dosificación , Animales , Análisis Heterodúplex , Humanos , Oligonucleótidos/química , ARN Catalítico/genética , ARN Mensajero/química , Ribonucleasa H/farmacologíaRESUMEN
Some regions of nucleic acid targets are not accessible to heteroduplex formation with complementary oligonucleotide probes because they are involved in secondary structure through intramolecular Watson-Crick pairing. The secondary conformation of the target may be destabilised to assist its interaction with oligonucleotide probes. To achieve this, we modified a DNA target, which has self-complementary sequence able to form a hairpin loop, by replacing dC with N:4-ethyldeoxycytidine (d(4Et)C), which hybridises specifically with natural dG to give a G:(4Et)C base pair with reduced stability compared to the natural G:C base pair. Substitution by d(4Et)C greatly reduced formation of the target secondary structure. The lower level of secondary structure allowed hybridisation with complementary probes made with natural bases. We confirmed that hybridisation could be further enhanced by modifying the probes with intercalating groups which stabilise the duplex.
Asunto(s)
Sondas de ADN/química , Sondas de ADN/metabolismo , ADN/química , ADN/metabolismo , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico/métodos , Emparejamiento Base/efectos de los fármacos , Secuencia de Bases , ADN/efectos de los fármacos , ADN/genética , Sondas de ADN/efectos de los fármacos , Sondas de ADN/genética , Desoxirribonucleósidos/química , Desoxirribonucleósidos/genética , Desoxirribonucleósidos/metabolismo , Ingeniería Genética , Sustancias Intercalantes/farmacología , Mutación/genética , Conformación de Ácido Nucleico/efectos de los fármacos , Desnaturalización de Ácido Nucleico/efectos de los fármacos , Desnaturalización de Ácido Nucleico/genética , Hibridación de Ácido Nucleico/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Temperatura , TermodinámicaRESUMEN
We describe the characterisation of four thermo-stable NAD(+)-dependent DNA ligases, from Thermus thermophilus (Tth), Thermus scotoductus (Ts), Rhodothermus marinus (Rm) and Thermus aquaticus (Taq), by an assay which measures ligation rate and mismatch discrimination. Complete libraries of octa-, nona- and decanucleotides were used as substrates. The assay comprised the polymerisation of oligo-nucleotides initiated from a 17 base 'primer', using M13mp18 ssDNA as template. Polymers of ligation products were analysed by polyacrylamide gel electro-phoresis. Under optimum conditions, the enzymes produced polymers ranging from 8 to 16 additions; there was variation between enzymes and the length of the oligonucleotides had a strong effect. The optimal total oligonucleotide concentration for each library was approximately 4 nmol. We compared the rates of ligation between the four ligases using an octanucleotide library as substrate. By this criterion, the Ts and Rm ligases are far more active compared to the more commonly available thermostable ligases.
Asunto(s)
Chlorobi/enzimología , ADN Ligasas/metabolismo , Thermus/enzimología , Cartilla de ADN , ADN de Cadena Simple/metabolismo , ADN Viral/metabolismo , Biblioteca de Genes , Oligodesoxirribonucleótidos/metabolismo , Especificidad por SustratoRESUMEN
The large-scale typing of sequence variation in genes and genomic DNA presents new challenges for which it is not clear that current technologies are sufficiently sensitive, robust, or scalable. This review surveys the current platform technologies: separation-based approaches, which include mass spectrometry; homogeneous assays; and solid-phase/array-based assays. We assess techniques for discovering and typing variation on a large scale, especially that of single-nucleotide polymorphisms. The in-depth focus is the DNA chip/array platform, and some of the published large-scale studies are closely examined. The problem of large-scale amplification is addressed, and emerging technologies for present and future needs are indicated.
Asunto(s)
ADN/genética , Variación Genética , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Genotipo , Globinas/genética , Haplotipos , Humanos , Espectrometría de Masas , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido SimpleRESUMEN
Despite the simplicity of the concept, almost every step in an antisense experiment poses difficulties. Finding a site that is accessible to intermolecular hybridization with complementary nucleic acids is a major problem and determines the success or failure of an antisense experiment. A major determinant of accessibility appears to be the intramolecular folding in mRNAs that renders much of the molecule inaccessible. However, owing to our poor understanding of RNA folding and the mechanisms of heteroduplex formation, theoretical methods have limited use in finding accessible sites. Such methods are unable to address two major considerations when designing an antisense reagent, i.e., which is the most accessible sequence in the target and what length of the reagent works best in terms of activity and specificity. Empirical approaches appear more successful. Of notable interest, and reviewed here, are 'global' methods based on DNA arrays and on mapping of transcripts with RNase H.
Asunto(s)
Hibridación de Ácido Nucleico , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , ARN/genética , ARN/metabolismo , Animales , Sitios de Unión/genética , Humanos , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Oligonucleótidos Antisentido/uso terapéutico , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
DNA dendrimers with two, three, six, nine or 27 arms were reassociated as complementary pairs in solution or with an array of complementary oligonucleotides on a solid support. In all cases, duplex stabilities were greater than those of unbranched molecules of equal length. A theoretical treatment for the process of dissociation of dendrimers explains the major properties of the complexes. The favourable features of DNA dendrimers-their enhanced stability and the simple predictability of their association behaviour-makes them promising as building blocks for the 'bottom up' approach to nano-assembly. These features also suggest applications in oligonucleotide array/DNA chip technology when higher hybridisation temperatures are required, for example, to melt secon-dary structure in the target.
Asunto(s)
Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Secuencia de Bases , Diseño de Fármacos , Cinética , Modelos Moleculares , Modelos Teóricos , Peso Molecular , Hibridación de Ácido Nucleico , Oligodesoxirribonucleótidos/genética , Temperatura , TermodinámicaRESUMEN
We have studied the effects of structure on nucleic acid heteroduplex formation by analyzing hybridization of tRNAphe to a complete set of complementary oligonucleotides, ranging from single nucleotides to dodecanucleotides. The analysis points to features in tRNA that determine heteroduplex yield. All heteroduplexes that give high yield include both double-stranded stems as well as single-stranded regions. Bases in the single-stranded regions are stacked onto the stems, and heteroduplexes terminate at potential interfaces for coaxial stacking. Heteroduplex formation is disfavored by sharp turns or a lack of helical order in single-stranded regions, competition from bases displaced from a stem, and stable tertiary interactions. The study is relevant to duplex formation on oligonucleotide microarrays and to antisense technologies.
Asunto(s)
Oligonucleótidos/química , ARN de Transferencia de Fenilalanina/química , Secuencia de Bases , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex , Hibridación de Ácido NucleicoRESUMEN
Folding pathways of large RNAs are poorly understood. We have addressed this question by hybridizing in vitro transcripts, which varied in size, to an array of antisense oligonucleotides. All transcripts included a common sequence and all but one shared the same start-point; the other had a small deletion of the 5' end. Minimal free energy calculations predicted quite different folds for these transcripts. However, hybridization to the array showed predominant features that were shared by transcripts of all lengths, though some oligonucleotides that hybridized strongly to the short transcripts gave weak interaction with longer transcripts. A full-length RNA fragment that had been denatured by heating and allowed to cool slowly gave the same hybridization result as a shorter transcript. Taken together, these results support theories that RNA folding creates local stable states that are trapped early in the transcription or folding process. As the transcript elongates, interactions are added between regions that are transcribed early and those transcribed late. The method here described helps in identifying regions in the transcripts that take part in long-range interactions.
Asunto(s)
Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos Antisentido/metabolismo , ARN/química , Calor , Sustancias Macromoleculares , Desnaturalización de Ácido Nucleico , Plásmidos/genética , Reacción en Cadena de la Polimerasa , ARN/metabolismoRESUMEN
Combinatorial libraries of oligonucleotides on beads were synthesised by a split-and-mix strategy using 5'-DMTr- or 5'-Fmoc- nucleoside phosphoramidites. Trityl moieties with different masses were used to encode for the bases coupled at each step in the synthesis of oligonucleotides selected by hybridisation from the libraries. Tags orthogonal to the nucleotides added were produced by coupling amines of different MW to an activated carboxyl group(s) on the trityl moiety. Tags can be released from the support by laser irradiation and measured directly by TOF without matrix. Alternatively, they may be released by an acidic treatment and then analysed by (MA)LDI-TOF.
Asunto(s)
Técnicas Químicas Combinatorias , Biblioteca de Genes , Oligodesoxirribonucleótidos/síntesis química , Indicadores y Reactivos , Oligodesoxirribonucleótidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Compuestos de TritiloRESUMEN
Complete libraries of oligonucleotides were used as substrates for Thermus thermophilus DNA ligase, on a M13mp18 ssDNA template. A 17mer primer was used to start a polymerisation process. Ladders of ligation products were analysed by gel electrophoresis. Octa-, nona- and decanucleotide libraries were compared. Nonanucleotides were optimum for polymerisation and up to 15 monomers were ligated. The fidelity of incorporation was studied by sequencing 28 clones (2268 bases) of nonanucleotide polymers, 12 monomers in length. Of the ligated monomers, 79% were the correct complementary sequence. In a total of 57 (2.5%) mispaired bases, there was a strong bias to G.T, G.A, G.G and A.G mismatches. Of the mismatches, 86% were found to be purines on the incoming oligonucleotide, of which 71% were G. There is evidence for clustering of mismatches within specific 9mers and at specific positions within these 9mers. The most frequent mismatches were at the 5'-terminus of the oligonucleotide, followed by the central position. We suggest that sequence selection was imposed by the ligase and not just by base pairing interactions. The ligase directs polymerisation in the 3' to 5' direction which we propose is linked to its role in lagging strand DNA replication.
Asunto(s)
ADN Ligasas/metabolismo , Cartilla de ADN/química , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Thermus thermophilus/enzimología , Bacteriófago M13/química , Emparejamiento Base , Secuencia de Bases , Cartilla de ADN/síntesis química , Cartilla de ADN/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Bases de Datos Factuales , Datos de Secuencia Molecular , Moldes GenéticosRESUMEN
The large amount of DNA sequence information produced in recent years has created a need for high-throughput methods in biology and genetics. These include sequencing, comparing gene sequences and genotyping. DNA arrays promise a highly parallel means for analysis of DNA that is fast and cost-effective, and offers scope for application to complex systems and processes. Recent years have seen continued transfer of technology from the microelectronics industry. Rapid application of the technology to genotyping, antisense oligonucleotide selection and gene expression analysis has illustrated the general power of this approach.
Asunto(s)
Secuencia de Bases , ADN/química , ADN/genética , Técnicas Genéticas , Electrónica , Expresión Génica , Genotipo , Miniaturización , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/química , Reacción en Cadena de la Polimerasa/métodos , Homología de Secuencia de Ácido NucleicoRESUMEN
The requirement for Watson-Crick base pairing surrounding a nick in duplex DNA to be sealed by DNA ligase is the basis for oligonucleotide ligation assays that distinguish single base mutations in DNA targets. Experiments in a model system demonstrate that the minimum length of oligonucleotide that can be joined differs for different ligases. Thermus thermophilus (Tth) DNA ligase is unable to join any oligonucleotide of length six or less, while T4 DNA ligase and T7 DNA ligase are both able to join hexamers. The rate of oligonucleotide ligation by Tth DNA ligase increases between heptamer and nonamer. Mismatches which cause the duplex to be shortened by fraying, at the end distal to the join, slow the ligation reaction. In the case of Tth DNA ligase, mismatches at the seventh and eighth position 5'to the nick completely inhibit the ligation of octamers. The results are relevant to mechanisms of ligation.
Asunto(s)
Composición de Base , ADN Ligasas/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Bacteriófago T7/enzimología , Secuencia de Bases , Geobacillus stearothermophilus/enzimología , Oligodesoxirribonucleótidos/química , Relación Estructura-Actividad , Especificidad por Sustrato , Temperatura , Thermus thermophilus/enzimologíaRESUMEN
An array of 1,938 oligodeoxynucleotides (ONs) ranging in length from monomers to 17-mers was fabricated on the surface of a glass plate and used to measure the potential of oligonucleotide for heteroduplex formation with rabbit beta-globin mRNA. The oligonucleotides were complementary to the first 122 bases of mRNA comprising the 5' UTR and bases 1 to 69 of the first exon. Surprisingly few oligonucleotides gave significant heteroduplex yield. Antisense activity, measured in a RNase H assay and by in vitro translation, correlated well with yield of heteroduplex on the array. These results help to explain the variable success that is commonly experienced in the choice of antisense oligonucleotides. For the optimal ON, the concentration required to inhibit translation by 50% was found to be five times less than for any other ON. We find no obvious features in the mRNA sequence or the predicted secondary structure that can explain the variation in heteroduplex yield. However, the arrays provide a simple empirical method of selecting effective antisense oligonucleotides for any RNA target of known sequence.
Asunto(s)
Globinas/biosíntesis , Oligodesoxirribonucleótidos/síntesis química , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Exones , Globinas/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/metabolismo , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Conejos , Transcripción GenéticaRESUMEN
We have investigated the use of spacer molecules to reduce steric interference of the support on the hybridisation behaviour of immobilised oligonucleotides. These spacers are built up from a variety of monomeric units, using phosphoramidite chemistry, by condensation onto an amine-functionalised polypropylene support. The optimal spacer length was determined to be at least 40 atoms in length, giving up to 150-fold increase in the yield of hybridisation. The effects of different charged groups in the spacer were also examined, and it was shown that both positively and negatively charged groups in the spacer diminish the yield of hybridisation. Steric hindrance in hybridisation can also be a problem if the oligonucleotides attached to the support are too close to each other. Surface coverage was varied using a combination of cleavable and stable linkers, giving the highest hybridisation yields for surfaces containing approximately 50% of the maximum concentration of oligonucleotides.