RESUMEN
We present a new design of mixed-backbone antisense oligonucleotides (ASOs) containing both DNA and peptide nucleic acid (PNA). Previous generations of PNA-DNA chimeras showed low binding affinity, reducing their potential as therapeutics. The addition of a 5'-wing of locked nucleic acid as well as the combination of a modified nucleotide and a PNA monomer at the junction between PNA and DNA yielded high-affinity chimeras. The resulting ASOs demonstrated high serum stability and elicited robust RNase H-mediated cleavage of complementary RNA. These properties allowed the chimeric ASOs to demonstrate high gene silencing efficacy and potency in cells, comparable with those of LNA gapmer ASOs, via both lipid transfection and gymnosis.
Asunto(s)
Silenciador del Gen , Oligonucleótidos Antisentido/farmacología , Ácidos Nucleicos de Péptidos/farmacología , ARN Largo no Codificante/antagonistas & inhibidores , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Oligonucleótidos Antisentido/química , Ácidos Nucleicos de Péptidos/química , ARN Largo no Codificante/genética , Ribonucleasa H/metabolismoRESUMEN
Reliable detection and quantification of antisense oligonucleotides (ASOs) in experimental and clinical specimens are essential to understand the biological function of novel oligonucleotide-based therapeutics. In this study, we describe a method to detect and quantify ASOs in biological samples, whereby the ASO acts as a splint to direct the ligation of complementary probes and quantitative real-time PCR was used to monitor ligation products. Low levels of 2'-O-methoxyethyl (2'-O-MOE) gapmer ASO in serum, liver, kidney, lung, heart, muscle, and brain tissues can be detected over a 6-log linear range for detection using this method. This method allows quantification of various types of chemically modified ASOs, including phosphorothioate linkage, 2'-O-methyl, 2'-O-MOE, and locked nucleic acid, as well as siRNAs. This method does not require probe modifications, and can be performed using standard laboratory equipment; making it a fast, sensitive, and reliable technique that can be widely applied. This detection method may find potential applications in detection of therapeutic oligonucleotides in biological samples.