RESUMEN
Targeted extrahepatic delivery of siRNA remains a challenging task in the field of nucleic acid therapeutics. An ideal delivery tool must internalize siRNA exclusively into the cells of interest without affecting the silencing activity of siRNA. Here, we report the use of anti-EGFR Nanobodies (trademark of Ablynx N.V.) as tools for targeted siRNA delivery. A straightforward procedure for site-specific conjugation of siRNA to an engineered C-terminal cysteine residue on the Nanobody (trademark of Ablynx N.V.) is described. We show that siRNA-conjugated Nanobodies (Nb-siRNA) retain their binding to EGFR and enter EGFR-positive cells via receptor-mediated endocytosis. The activity of Nb-siRNAs was assessed by measuring the knockdown of a housekeeping gene (AHSA1) in EGFR-positive and EGFR-negative cells. We demonstrate that Nb-siRNAs are active in vitro and induce mRNA cleavage in the targeted cell line. In addition, we discuss the silencing activity of siRNA conjugated to fused Nbs with various combinations of EGFR-binding building blocks. Finally, we compare the performance of Nb-siRNA joined by four different linkers and discuss the advantages and limitations of using cleavable and noncleavable linkers in the context of Nanobody-mediated siRNA delivery.
Asunto(s)
Neoplasias/genética , Neoplasias/terapia , ARN Interferente Pequeño/genética , Anticuerpos de Dominio Único/genética , Línea Celular Tumoral , Receptores ErbB/genética , Silenciador del Gen/fisiología , Células Hep G2 , Humanos , Ácidos Nucleicos/genéticaRESUMEN
Detection of pathogenic nucleic acids remains one of the most reliable approaches for the diagnosis of a broad range of diseases. Current PCR-based methods require experienced personnel and cannot be easily used for point-of-care diagnostics, making alternative strategies for the sensitive, reliable, and cost-efficient detection of pathogenic nucleic acids highly desirable. Here, we report an enzyme-free method for the fluorometric detection of RNA that relies on a target-induced fluorophore transfer onto a semiconductor quantum dot (QD), uses PNA probes as selective recognition elements and can be read out with simple and inexpensive equipment. For QD-PNA conjugates with optimized PNA content, limits of detection of dengue RNA in the range of 10 pM to 100 nM can be realized within 5 h in the presence of a high excess of noncomplementary RNA.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Hibridación de Ácido Nucleico/métodos , Ácidos Nucleicos de Péptidos/química , Puntos Cuánticos/química , ARN/análisis , Dengue/diagnóstico , Virus del Dengue/aislamiento & purificación , Humanos , ARN Viral/análisisRESUMEN
Oligonucleotide-templated reactions (OTRs) between two reactive hybridization probes allow for the detection of a DNA or RNA of interest by exploiting the target molecule as a catalyst of chemical reactions. The product of such a reaction commonly exhibits distinct fluorescence properties and can be detected by the means of fluorescence spectroscopy. The vast majority of OTR systems utilize organic dyes as fluorescent reporters. However, the use of brighter emitters, such as semiconductor quantum dots (QDs), has potential to improve the sensitivity of detection by providing brighter signals and permitting the use of probes at very low concentrations. Here we report an RNA-templated reaction between two fluorescently labeled peptide nucleic acid (PNA)-based probes, which proceeds on the surface of a QD. The QD-bound PNA probe bears a cysteine functionality, while the other PNA is functionalized with an organic dye as a thioester. OTR between these probes proceeds through a transfer of the organic dye to the QD and can be conveniently monitored via fluorescence resonance energy transfer (FRET) from the QD to the Cy5. The reaction was performed in a conventional fluorescence microplate reader and permits the detection of RNA in the picomolar range.