RESUMEN
Most human pre-mRNAs are cis-spliced, removing introns and joining flanking exons of the same RNA molecule. However, splicing of exons present on separate pre-mRNA molecules can also occur. This trans-splicing reaction can be exploited by pre-trans-splicing molecules (PTMs), which are incapable of cis-splicing. PTM-mediated trans-splicing has been utilized to repair mutant RNAs as a novel approach to gene therapy. Herein we explore how the site of PTM expression influences trans-splicing activity. We stably inserted a PTM expression cassette into the genome of HEK293 cells, generating clonal lines with single, unique insertion sites. We analyzed trans-splicing to the gene where the PTM was integrated, as well as genes neighboring these loci. We observed some pre-mRNAs only serve as substrates for trans-splicing when they are expressed in immediate proximity to the PTM expression site. The need for PTMs to be in close proximity with pre-mRNAs to trans-splice with them is consistent with the observation that pre-mRNA cis-splicing occurs cotranscriptionally. Interestingly, we identified several cellular pre-mRNAs in one localized area that serve as trans-splicing substrates irrespective of the PTM expression site. Thus, we find multiple cellular pre-mRNAs require PTM expression in close proximity to trans-splice while others do not.
Asunto(s)
Precursores del ARN/metabolismo , Trans-Empalme , Transcripción Genética , Animales , Línea Celular , Exones , Humanos , Ratas , EmpalmosomasRESUMEN
Technologies that mediate targeted delivery of small interfering RNAs (siRNAs) are needed to improve their therapeutic efficacy and safety. Therefore, we have developed aptamer-siRNA chimeric RNAs capable of cell type-specific binding and delivery of functional siRNAs into cells. The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. When applied to cells expressing PSMA, these RNAs are internalized and processed by Dicer, resulting in depletion of the siRNA target proteins and cell death. In contrast, the chimeras do not bind to or function in cells that do not express PSMA. These reagents also specifically inhibit tumor growth and mediate tumor regression in a xenograft model of prostate cancer. These studies demonstrate an approach for targeted delivery of siRNAs with numerous potential applications, including cancer therapeutics.
Asunto(s)
Aptámeros de Nucleótidos/genética , Silenciador del Gen , Marcación de Gen/métodos , Técnicas de Transferencia de Gen , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Animales , Aptámeros de Nucleótidos/administración & dosificación , Línea Celular Tumoral , HumanosRESUMEN
Tie2 is a receptor tyrosine kinase that is expressed predominantly in the endothelium and plays key roles in both physiological and pathological angiogenesis. The ligands for Tie2, the angiopoietins (Ang), perform opposing functions in vascular maintenance and angiogenesis; Ang1 regulates vascular quiescence, while Ang2 is thought to promote vascular destabilization and facilitate angiogenesis. However, the mechanisms responsible for these differences are not understood. To begin to elucidate the molecular differences between the angiopoietins, we previously developed a specific RNA aptamer inhibitor of Ang2. Here, we used the same iterative in vitro selection process, termed SELEX (Systematic Evolution of Ligands by EXponential enrichment), to screen a library of 2'-fluoro-modified ribonucleotides for Ang1-binding aptamers. After nine rounds of selection, we identified a single clone, ANG9-4, that bound with high affinity to human Ang1 (K ( d ) 2.8 nM) but not Ang2 (K ( d ) > 1 microM), demonstrating specificity for Ang1. ANG9-4 blocked Ang1-mediated Tie2 phosphorylation and downstream Akt activation. Moreover, ANG9-4 inhibited Ang1-induced endothelial cell survival. Together, these findings demonstrate the feasibility of developing an Ang1-inhibitory aptamer. ANG9-4 and its derivatives may provide useful tools for elucidating the biology of Ang1 and for treating certain angiogenic diseases.
Asunto(s)
Angiopoyetina 1/farmacología , Aptámeros de Nucleótidos/farmacología , Endonucleasas/metabolismo , Receptor TIE-2/antagonistas & inhibidores , Receptor TIE-2/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Activación Enzimática/efectos de los fármacos , Humanos , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Venas Umbilicales/citologíaRESUMEN
We investigated the T locus as a candidate gene in a series of patients and families with lumbosacral myelomeningocele. Single-strand conformation polymorphism (SSCP) analysis was used to identify sequence variation in all 8 exons and in intron 7 of this locus. We found evidence of substantial polymorphism within this locus, as previously reported [Papapetrou et al., 1999, J Med Genet 36:208-213], and moderately significant evidence of linkage disequilibrium with the CacI polymorphism of exon 8. However, when the locus was considered as a whole, with all single nucleotide polymorphisms (SNPs) integrated into a haplotype, there was no evidence for linkage disequilibrium. In addition, we did not identify any new sequence variants. Thus, we conclude that the T locus is not a major locus for human NTDs in this sample.
Asunto(s)
Proteínas Fetales , Defectos del Tubo Neural/genética , Proteínas de Dominio T Box/genética , Población Blanca/genética , Alelos , Sustitución de Aminoácidos , ADN/química , ADN/genética , Análisis Mutacional de ADN , Salud de la Familia , Frecuencia de los Genes , Humanos , Desequilibrio de Ligamiento , Mutación , Polimorfismo de Nucleótido Simple , Polimorfismo Conformacional Retorcido-Simple , Estados UnidosRESUMEN
Aptamers are short, single-stranded oligonucleotides that are isolated through a process termed systematic evolution of ligands by exponential enrichment. With the advent of cell-based selection technology, aptamers can be selected to bind protein targets that are expressed on the cell surface. These aptamers demonstrate excellent specificity and high affinity toward their target proteins and are often internalized upon binding to their targets. This has opened up the possibility of using aptamers for cell-specific targeted drug delivery. In this review, we will discuss cell-surface protein targets, the aptamers that bind them, and their applications for targeted therapeutics.