RESUMEN
Antisense oligonucleotides are used for therapeutic applications and in functional genomic studies. In practice, however, many of the oligonucleotides complementary to an mRNA have little or no antisense activity. Theoretical strategies to improve the 'hit rate' in antisense screens will reduce the cost of discovery and may lead to identification of antisense oligonucleotides with increased potency. Statistical analysis performed on data collected from more than 1000 experiments with phosphorothioate-modified oligonucleotides revealed that the oligo-probes, which form stable duplexes with RNA (DeltaG(o)37 < or = -30 kcal/mol) and have small self-interaction potential, are more frequently efficient than molecules that form less stable oligonucleotide-RNA hybrids or more stable self-structures. To achieve optimal statistical preference, the values for self-interaction should be (DeltaG(o)37) > or = -8 kcal/mol for inter-oligonucleotide pairing and (DeltaG(o)37) > or = -1.1 kcal/mol for intra-molecular pairing. Selection of oligonucleotides with these thermodynamic values in the analyzed experiments would have increased the 'hit rate' by as much as 6-fold.
Asunto(s)
Oligonucleótidos Antisentido/química , Termodinámica , Química Farmacéutica/métodos , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/química , ARN Mensajero/genética , Sensibilidad y EspecificidadRESUMEN
RNA multibranch loops (junctions) are loops from which three or more helices exit. They are nearly ubiquitous in RNA secondary structures determined by comparative sequence analysis. In this study, systems in which two strands combine to form three-way junctions were used to measure the stabilities of RNA multibranch loops by UV optical melting and isothermal titration calorimetry (ITC). These data were used to calculate the free energy increment for initiation of a three-way junction on the basis of a nearest neighbor model for secondary structure stability. Imino proton NMR spectra were also measured for two systems and are consistent with the hypothesized helical structures. Incorporation of the experimental data into the mfold and RNA structure computer programs has contributed to an improvement in prediction of RNA secondary structure from sequence.
Asunto(s)
Conformación de Ácido Nucleico , ARN/química , Bacteriófago T7/enzimología , Calorimetría , ARN Polimerasas Dirigidas por ADN/genética , Magnesio/química , Resonancia Magnética Nuclear Biomolecular , Desnaturalización de Ácido Nucleico , Oligonucleótidos/química , ARN/síntesis química , ARN/genética , ARN Ribosómico 5S/química , Moldes Genéticos , Termodinámica , Proteínas ViralesRESUMEN
A computer program, OligoWalk, is reported that predicts the equilibrium affinity of complementary DNA or RNA oligonucleotides to an RNA target. This program considers the predicted stability of the oligonucleotide-target helix and the competition with predicted secondary structure of both the target and the oligonucleotide. Both unimolecular and bimolecular oligonucleotide self structure are considered with a user-defined concentration. The application of OligoWalk is illustrated with three comparisons to experimental results drawn from the literature.
Asunto(s)
Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligorribonucleótidos/química , ARN/química , Programas Informáticos , Secuencia de Bases , Sitios de Unión , Calorimetría , ADN Complementario/química , Globinas/genética , Cinética , Modelos Teóricos , Datos de Secuencia Molecular , ARN Complementario/química , ARN Mensajero/química , ARN Mensajero/genética , Ribonucleasa H , TermodinámicaRESUMEN
An improved dynamic programming algorithm is reported for RNA secondary structure prediction by free energy minimization. Thermodynamic parameters for the stabilities of secondary structure motifs are revised to include expanded sequence dependence as revealed by recent experiments. Additional algorithmic improvements include reduced search time and storage for multibranch loop free energies and improved imposition of folding constraints. An extended database of 151,503 nt in 955 structures? determined by comparative sequence analysis was assembled to allow optimization of parameters not based on experiments and to test the accuracy of the algorithm. On average, the predicted lowest free energy structure contains 73 % of known base-pairs when domains of fewer than 700 nt are folded; this compares with 64 % accuracy for previous versions of the algorithm and parameters. For a given sequence, a set of 750 generated structures contains one structure that, on average, has 86 % of known base-pairs. Experimental constraints, derived from enzymatic and flavin mononucleotide cleavage, improve the accuracy of structure predictions.
Asunto(s)
Secuencia de Aminoácidos , Conformación de Ácido Nucleico , Estructura Secundaria de Proteína , Termodinámica , Algoritmos , Bacteriófago T4/química , Bases de Datos Factuales , Escherichia coli/química , Mononucleótido de Flavina/farmacología , Cinética , Modelos Genéticos , Modelos Estadísticos , Datos de Secuencia Molecular , ARN Ribosómico 5S/química , Factores de TiempoRESUMEN
RNA transcripts corresponding to the 250-nt 3' untranslated region of the R2 non-LTR retrotransposable element are recognized by the R2 reverse transcriptase and are sufficient to serve as templates in the target DNA-primed reverse transcription (TPRT) reaction. The R2 protein encoded by the Bombyx mori R2 can recognize this region from both the B. mori and Drosophila melanogaster R2 elements even though these regions show little nucleotide sequence identity. A model for the RNA secondary structure of the 3' untranslated region of the D. melanogaster R2 retrotransposon was developed by sequence comparison of 10 species aided by free energy minimization. Chemical modification experiments are consistent with this prediction. A secondary structure model for the 3' untranslated region of R2 RNA from the R2 element from B. mori was obtained by a combination of chemical modification data and free energy minimization. These two secondary structure models, found independently, share several common sites. This study shows the utility of combining free energy minimization, sequence comparison, and chemical modification to model an RNA secondary structure.
Asunto(s)
Conformación de Ácido Nucleico , ADN Polimerasa Dirigida por ARN/metabolismo , ARN/química , Retroelementos/genética , Algoritmos , Animales , Secuencia de Bases , Bombyx/genética , Simulación por Computador , Drosophila/clasificación , Drosophila/genética , Metilación , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico , Unión Proteica , ARN/efectos de los fármacos , ARN/metabolismo , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Especificidad por Sustrato , Temperatura , Moldes Genéticos , TermodinámicaRESUMEN
An RNA model system consisting of an oligomer binding to a 4-nt overhang at the 5' end of a hairpin stem provides thermodynamic parameters for helix-helix interfaces. In a sequence-dependent manner, oligomers bind up to 1000-fold more tightly adjacent to the hairpin stem than predicted for binding to a free tetramer at 37 degrees C. For the interface (/) in [formula: see text] additional free energy change, delta delta G 37 degrees, for binding is roughly the nearest-neighbor delta G 37 degrees for propagation of an uninterrupted helix of equivalent sequence, CGGC. When X and Z are omitted, the delta delta 37 degrees is even more favorable by approximately 1 kcal/mol (1 cal = 4.184J). On average, predictions of 11 RNA secondary structures improve from 67 to 74% accuracy by inclusion of similar stacking contributions.