RESUMO
The syntheses of 10 new RNA 2'-O-modifications, their incorporation into oligonucleotides, and an evaluation of their properties such as RNA affinity and nuclease resistance relevant to antisense activity are presented. All modifications combined with the natural phosphate backbone lead to significant gains in terms of the stability of hybridization to RNA relative to the first-generation DNA phosphorothioates (PS-DNA). The nuclease resistance afforded in particular by the 2'-O-modifications carrying a positive charge surpasses that of PS-DNA. However, small electronegative 2'-O-substituents, while enhancing the RNA affinity, do not sufficiently protect against degradation by nucleases. Similarly, oligonucleotides containing 3'-terminal residues modified with the relatively large 2'-O-[2-(benzyloxy)ethyl] substituent are rapidly degraded by exonucleases, proving wrong the assumption that steric bulk will generally improve protection against nuclease digestion. To analyze the factors that contribute to the enhanced RNA affinity and nuclease resistance we determined crystal structures of self-complementary A-form DNA decamer duplexes containing single 2'-O-modified thymidines per strand. Conformational preorganization of substituents, favorable electrostatic interactions between substituent and sugar-phosphate backbone, and a stable water structure in the vicinity of the 2'-O-modification all appear to contribute to the improved RNA affinity. Close association of positively charged substituents and phosphate groups was observed in the structures with modifications that protect most effectively against nucleases. The promising properties exhibited by some of the analyzed 2'-O-modifications may warrant a more detailed evaluation of their potential for in vivo antisense applications. Chemical modification of RNA can also be expected to significantly improve the efficacy of small interfering RNAs (siRNA). Therefore, the 2'-O-modifications introduced here may benefit the development of RNAi therapeutics.
Assuntos
Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , RNA/química , RNA/metabolismo , Ribonucleases/metabolismo , Sequência de Bases , Fenômenos Biofísicos , Biofísica , Cristalização , Cristalografia por Raios X , DNA/química , DNA/genética , DNA/metabolismo , Estabilidade Enzimática , Exonucleases/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Oligonucleotídeos/genética , RNA/genética , Eletricidade Estática , TemperaturaRESUMO
A combination of algorithms to search RNA sequence for the potential for secondary structure formation, and search large numbers of sequences for structural similarity, were used to search the 5'UTRs of annotated genes in the Escherichia coli genome for regulatory RNA structures. Using this approach, similar RNA structures that regulate genes in the thiamin metabolic pathway were identified. In addition, several putative regulatory structures were discovered upstream of genes involved in other metabolic pathways including glycerol metabolism and ethanol fermentation. The results demonstrate that this computational approach is a powerful tool for discovery of important RNA structures within prokaryotic organisms.
Assuntos
Regiões 5' não Traduzidas/genética , Mapeamento Cromossômico/métodos , Escherichia coli/genética , Regulação da Expressão Gênica/genética , Alinhamento de Sequência/métodos , Análise de Sequência de RNA/métodos , Transdução de Sinais/genética , Sequência de Bases , Sequência Conservada , Dados de Sequência MolecularRESUMO
[structure: see text] Oligonucleotides with a novel 2'-O-[2-(guanidinium)ethyl] (2'-O-GE) modification have been synthesized using a novel protecting group strategy for the guanidinium group. This modification enhances the binding affinity of oligonucleotides to RNA as well as duplex DNA (DeltaT(m) 3.2 degrees C per modification). The 2'-O-GE modified oligonucleotides exhibited exceptional resistance to nuclease degradation. The crystal structure of a palindromic duplex formed by a DNA oligonucleotide with a single 2'-O-GE modification was solved at 1.16 A resolution.
Assuntos
DNA/química , Guanidina/química , Oligonucleotídeos/química , RNA/química , DNA Complementar/química , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos/síntese química , RNA Complementar/química , Fatores de TempoRESUMO
A phosphorothioate-linked oligonucleotide bearing a 3'-terminal phosphorothioate monoester has been synthesized and characterized. This oligonucleotide has been identified as a process-related impurity formed during synthesis of ISIS 2302. Biological properties of the compound have been determined. Based on these data, it can be concluded that this species (3'-TPT) has biological properties similar to parent drug.
Assuntos
Molécula 1 de Adesão Intercelular/genética , Oligodesoxirribonucleotídeos Antissenso/síntese química , Tionucleotídeos/síntese química , Animais , Tecnologia Biomédica , Sistemas de Liberação de Medicamentos , Cromatografia Gasosa-Espectrometria de Massas , Imunossupressores/síntese química , Imunossupressores/isolamento & purificação , Imunossupressores/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Cinética , Oligodesoxirribonucleotídeos Antissenso/química , Oligodesoxirribonucleotídeos Antissenso/metabolismo , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/isolamento & purificação , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Fosforotioatos , Controle de Qualidade , Ribonuclease H/fisiologia , Tionucleotídeos/química , Tionucleotídeos/metabolismoRESUMO
Inclusion of C-5 propynyl pyrimidines in phosphorothioate antisense oligonucleotides (ASOs) has been shown to significantly increase their potency for inhibiting gene expression in vitro. This increased potency is believed to be the result of enhanced binding affinity to target RNA. Our results show that C-5 propynyl pyrimidine-modified oligonucleotides caused an increase in the melting temperature (T(m)) of both oligodeoxynucleotides (ODNs) and 2'-O-(2-methoxy)ethyl (2'-MOE)-modified oligonucleotides. The in vitro data show a moderate increase in potency for an antisense oligodeoxynucleotide containing C-5 propynyl pyrimidines targeting the murine PTEN (MMAC1) transcript. Second-generation 2'-MOE chimeric ASOs containing C-5 propynyl pyrimidines showed no improvement in potency in PTEN target reduction in vitro or in vivo compared to their nonpropyne-modified parent. These results suggest that increasing affinity for target RNA beyond that achieved with the 2'-MOE modification does not further increase potency in cell-based assays. To evaluate whether this observation held true for in vivo applications, we evaluated both compounds in mice. We were unable to establish a dose-response relationship with C-5 propynyl pyrimidine-modified ODNs because of severe toxicity. The toxicity was characterized by mortality in animals receiving 50 mg/kg and an increase in infiltrating cells and apoptotic cells in livers of mice receiving 20 mg/kg. C-5 propynyl pyrimidine-modified chimeric oligonucleotides exhibited decreased hepatotoxicity compared with C-5 propynyl-modified ODNs but did not exhibit an increase in potency compared with unmodified chimeric oligonucleotides. The hepatotoxicity could be further limited if incorporation of propynyl pyrimidines was restricted to 2'-MOE nucleosides.
Assuntos
Endotélio Vascular/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Nucleotídeos de Pirimidina/química , Alanina Transaminase/sangue , Animais , Apoptose/efeitos dos fármacos , Aspartato Aminotransferases/sangue , Sequência de Bases , Encéfalo/irrigação sanguínea , Linhagem Celular , Esquema de Medicação , Endotélio Vascular/efeitos dos fármacos , Injeções Intraperitoneais , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/toxicidade , Tamanho do Órgão/efeitos dos fármacos , RNA/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
[structure: see text] Oligonucleotides with novel modifications, 2'-O-[2-(amino)-2-oxoethyl] (2'-O-NAc), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMAc), 2'-O-[2-(dimethylamino)-2-oxoethyl] (2'-O-DMAc), and 2'-O-[2-[[2-(dimethylamino)ethyl]amino]-2-oxoethyl] (2'-O-DMAEAc), have been synthesized. These modified oligonucleotides exhibit high binding affinity to complementary RNA (and not to DNA) and considerably enhance the nuclease stability of oligonucleotides with t(1/2) > 24 h.
Assuntos
Oligonucleotídeos Antissenso/síntese química , Estabilidade de Medicamentos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , Fosfodiesterase I , Diester Fosfórico Hidrolases/metabolismo , RNA Complementar/metabolismo , Relação Estrutura-AtividadeRESUMO
[structure: see text] Peptide nucleic acid (PNA) monomers containing the tricyclic cytosine analogues phenoxazine, 9-(2-aminoethoxy)phenoxazine (G-clamp), and 9-(3-aminopropoxy)phenoxazine (propyl-G-clamp) have been synthesized. The modified nucleobases were incorporated into PNA oligomers using Boc-chemistry for solid-phase synthesis. PNAs containing single G-clamp modifications exhibit significantly enhanced affinity toward RNA and DNA targets relative to unmodified PNA while maintaining mismatch discrimination. These PNA G-clamp modifications exhibit the highest increase in affinity toward nucleic acid targets reported so far for PNA modifications.
Assuntos
Citosina/análogos & derivados , Citosina/química , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/síntese química , DNA/química , DNA/metabolismo , Estrutura Molecular , Oxazinas/química , RNA/química , RNA/metabolismoRESUMO
RNA molecules fold into characteristic secondary and tertiary structures that account for their diverse functional activities. Many of these RNA structures, or certain structural motifs within them, are thought to recur in multiple genes within a single organism or across the same gene in several organisms and provide a common regulatory mechanism. Search algorithms, such as RNAMotif, can be used to mine nucleotide sequence databases for these repeating motifs. RNAMotif allows users to capture essential features of known structures in detailed descriptors and can be used to identify, with high specificity, other similar motifs within the nucleotide database. However, when the descriptor constraints are relaxed to provide more flexibility, or when there is very little a priori information about hypothesized RNA structures, the number of motif 'hits' may become very large. Exhaustive methods to search for similar RNA structures over these large search spaces are likely to be computationally intractable. Here we describe a powerful new algorithm based on evolutionary computation to solve this problem. A series of experiments using ferritin IRE and SRP RNA stem-loop motifs were used to verify the method. We demonstrate that even when searching extremely large search spaces, of the order of 10(23) potential solutions, we could find the correct solution in a fraction of the time it would have taken for exhaustive comparisons.
Assuntos
Biologia Computacional/métodos , RNA/química , Sequências Reguladoras de Ácido Ribonucleico , Algoritmos , Animais , Sequência de Bases , Evolução Molecular , Ferritinas/genética , Humanos , Ferro/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Partícula de Reconhecimento de Sinal/químicaRESUMO
Lentiviruses (a sub-family of the retroviridae family) include primate and non-primate viruses associated with chronic diseases of the immune system and the central nervous system. All lentiviruses encode a regulatory protein Rev that is essential for post-transcriptional transport of the unspliced and incompletely spliced viral mRNAs from nuclei to cytoplasm. The Rev protein acts via binding to an RNA structural element known as the Rev responsive element (RRE). The RRE location and structure and the mechanism of the Rev-RRE interaction in primate and non-primate lentiviruses have been analyzed and compared. Based on structural data available for RRE of HIV-1, a two step computational strategy for prediction of putative RRE regions in lentivirus genomes has been developed. First, the RNAMotif algorithm was used to search genomic sequence for highly structured regions (HSR). Then the program RNAstructure, version 3.6 was used to calculate the structure and thermodynamic stability of the region of approximately 350 nucleotides encompassing the HSR. Our strategy correctly predicted the locations of all previously reported lentivirus RREs. We were able also to predict the locations and structures of potential RREs in four additional lentiviruses.
Assuntos
Algoritmos , Produtos do Gene rev , Lentivirus/genética , Elementos de Resposta , Animais , Biologia Computacional/métodos , HIV-1/genética , HIV-2/genética , Humanos , Primatas/virologia , Termodinâmica , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
A novel 2'-modification, 2'-O-[2-(methylthio)ethyl] or 2'-O-MTE, has been incorporated into oligonucleotides and evaluated for properties relevant to antisense activity. The results were compared with the previously characterized 2'-O-[2-(methoxy)ethyl] 2'-O-MOE modification. As expected, the 2'-O-MTE modified oligonucleotides exhibited improved binding to human serum albumin compared to the 2'-O-MOE modified oligonucleotides. The 2'-O-MTE oligonucleotides maintained high binding affinity to target RNA. Nuclease digestion of 2'-O-MTE oligonucleotides showed that they have limited resistance to exonuclease degradation. We analyzed the crystal structure of a decamer DNA duplex containing the 2'-O-MTE modifcation. Analysis of the crystal structure provides insight into the improved RNA binding affinity, protein binding affinity and limited resistance of 2'-O-MTE modified oligonucleotides to exonuclease degradation.
Assuntos
RNA/química , Uridina/análogos & derivados , Uridina/química , Sítios de Ligação , Cristalografia por Raios X , Ácidos Nucleicos Heteroduplexes/química , Nucleosídeos/química , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos Antissenso/química , Compostos Organofosforados/química , Ligação ProteicaRESUMO
The recent explosion in available bacterial genome sequences has initiated the need to improve an ability to annotate important sequence and structural elements in a fast, efficient and accurate manner. In particular, small non-coding RNAs (sRNAs) have been difficult to predict. The sRNAs play an important number of structural, catalytic and regulatory roles in the cell. Although a few groups have recently published prediction methods for annotating sRNAs in bacterial genome, much remains to be done in this field. Toward the goal of developing an efficient method for predicting unknown sRNA genes in the completed Escherichia coli genome, we adopted a bioinformatics approach to search for DNA regions that contain a sigma70 promoter within a short distance of a rho-independent terminator. Among a total of 227 candidate sRNA genes initially identified, 32 were previously described sRNAs, orphan tRNAs, and partial tRNA and rRNA operons. Fifty-one are mRNAs genes encoding annotated extremely small open reading frames (ORFs) following an acceptable ribosome binding site. One hundred forty-four are potentially novel non-translatable sRNA genes. Using total RNA isolated from E. coli MG1655 cells grown under four different conditions, we verified transcripts of some of the genes by Northern hybridization. Here we summarize our data and discuss the rules and advantages/disadvantages of using this approach in annotating sRNA genes on bacterial genomes.