Abstract
The use of short interfering RNAs (siRNAs) as therapeutics holds great promise, but chemical modifications must first be employed to improve their pharmacokinetic properties. This study evaluates the in vitro cellular uptake and knock-down efficacy of cholesterol-modified triazole-linked siRNAs targeting firefly luciferase in the absence of a transfection carrier. These siRNAs displayed low cytotoxicity and excellent dose-dependent knockdown in HeLa cells in the 500 to 3000 nM concentration range, with a 70–80% reduction in firefly luciferase activity. Our results indicate that this modification is compatible with the RNA interference pathway, and is less cytotoxic and more effective than a commercially-available triethylene glycol (TEG) cholesterol modification.
Highlights
The use of short interfering RNAs as therapeutics holds great promise, but chemical modifications must first be employed to improve their pharmacokinetic properties
Our results indicate that this modification is compatible with the RNA interference pathway, and is less cytotoxic and more effective than a commercially-available triethylene glycol (TEG) cholesterol modification
The initial step involves cleavage of long double-stranded RNA into smaller 21–23 nucleotide fragments, termed short interfering RNAs, which are incorporated into the RNA-induced silencing complex (RISC).[2]
Summary
RNA interference (RNAi) is an endogenous pathway that utilizes double-stranded RNA to suppress translation, resulting in sequence-speci c gene silencing.[1] The initial step involves cleavage of long double-stranded RNA into smaller 21–23 nucleotide fragments, termed short interfering RNAs (siRNAs), which are incorporated into the RNA-induced silencing complex (RISC).[2] RISC unwinds and dissociates the duplex, retaining the antisense strand which is used as a guiding sequence to recognize and degrade complementary mRNA.[2,3] Since many diseases are characterized by aberrant gene expression, the use of siRNAs as therapeutics holds great promise.[4,5] there are some limitations associated with the structure of siRNAs, including low stability, poor cellular uptake and off-target effects, which must be addressed in order to harness the full potential of RNAi therapeutics.[6,7] several chemical modi cations have been employed to improve the pharmacological properties of siRNAs, there is still no universal modi cation able to simultaneously improve all of these limitations.[8,9] Due to their large size and anionic backbone, siRNAs have difficulties crossing cellular membranes. Our biological studies in HeLa cells showed that these siRNAs were able to downregulate exogenous re y luciferase mRNA in a dose-
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