title: Optimized luciferase assay for cell-penetrating peptide-mediated delivery of short oligonucleotides.
abstract: An improved assay for screening for the intracellular delivery efficacy of short oligonucleotides using cell-penetrating peptides is suggested. This assay is an improvement over previous assays that use luciferase reporters for cell-penetrating peptides because it has been scaled up from a 24-well format to a 96-well format and no longer relies on a luciferin reagent that has been commercially sourced. In addition, the homemade luciferin reagent is useful in multiple cell lines and in different assays that rely on altering the expression of luciferase. To establish a new protocol, the composition of the luciferin reagent was optimized for both signal strength and longevity by multiple two-factorial experiments varying the concentrations of adenosine triphosphate, luciferin, coenzyme A, and dithiothreitol. In addition, the optimal conditions with respect to cell number and time of transfection for both short interfering RNA (siRNA) and splice-correcting oligonucleotides (SCOs) are established. Optimal transfection of siRNA and SCOs was achieved using the reverse transfection method where the oligonucleotide complexes are already present in the wells before the cells are plated. Z' scores were 0.73 for the siRNA assay and 0.71 for the SCO assay, indicating that both assays are suitable for high-throughput screening.
authors.affiliation: Department of Neurochemistry, Stockholm University, Stockholm 10691, Sweden. Electronic address: henrik.helmfors@neurochem.su.se.
authors.forename: Henrik
authors.initials: H
authors.lastname: Helmfors
authors.affiliation: Department of Neurochemistry, Stockholm University, Stockholm 10691, Sweden.
authors.forename: Jonas
authors.initials: J
authors.lastname: Eriksson
authors.affiliation: Department of Neurochemistry, Stockholm University, Stockholm 10691, Sweden.
authors.forename: Ülo
authors.initials: Ü
authors.lastname: Langel
mesh_terms: D001483:Base Sequence; D057846:Cell-Penetrating Peptides / Q000378:metabolism*; D003362:Cost-Benefit Analysis; D004337:Drug Carriers / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D049409:Luciferases, Firefly / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection / Q000191:economics / Q000379:methods*
chemical_list: D057846:Cell-Penetrating Peptides; D004337:Drug Carriers; D034741:RNA, Small Interfering; D049409:Luciferases, Firefly
keywords: Cell-penetrating peptide (CPP); High-throughput screening (HTS); Luciferase; Short interfering RNA (siRNA); Splice-correcting oligonucleotide (SCO)