Single cell kinetics of RNAi mediated knockdown

Abstract

Eukaryotic cells employ the RNA interference (RNAi) machinery in the cytosol to downregulate gene expression. Short 21-nucleotide microRNAs (miRNAs) associate with the so-called RNA induced silencing complex (RISC) and hybridize with the target gene’s mRNA, preventing it from being translated into protein. Employing the RNAi machinery with synthetic small interfering RNAs (siRNAs) that mimics the function of miRNAs, are currently a promising therapeutic approach for treatment of multiple diseases. However, reaching target tissues beyond the liver and getting macromolecular siRNAs into the cytosol of target cells are major issues. With current delivery strategies, only a minimal fraction of delivered siRNA is believed to reach the cytosol and the RNAi machinery, while the rest is trapped within the endosomal system. A key issue that complicates efforts to enhance the delivery efficiency of various siRNA delivery agents is a lack of tools to determine the absolute efficiency of a delivery strategy. In particular, the relationship between the amount of delivered siRNA and knockdown efficiency is unknown. Here, we set out to improve a previously established method for detection of small amounts of cytosolic siRNA molecules and to follow the resulting knockdown of a reporter gene, to ultimately determine the minimum number of siRNAs necessary for knockdown. A sensitive confocal microscopy setup was employed for live-cell imaging of HeLa cells to monitor cytosolic delivery of Alexa Fluor 647 labelled siRNA (siRNA-AF647) delivered with a widely used commercial transfection lipid. Cytosolic release events were detected automatically with a refined algorithm. Comparison to an independent method to detect siRNA delivery using a fluorescent membrane damage sensor (galectin-9) allowed determination of sensitivity and specificity of the detection algorithm. By a combination of automatic detection and manual quality control, a combined sensitivity and specificity of 89% and 99%, respectively, was achieved. Incubating HeLa cells stably expressing a short half-life eGFP protein with small amounts of siRNA-AF647 targeting eGFP demonstrated that 2 nM cytosolic siRNA concentration (~2000 molecules) resulted in complete knockdown while between 0.5 and 1.5 nM (~500-1500 molecules) resulted in varying degrees of knockdown, indicating that a minimum limit for complete knockdown is in the range of 1500-2000 molecules of siRNA for an siRNA with an IC50 of ~0.1nM. Additional experiments are currently being carried out with siRNA sequences with different IC50 values to determine how the IC50 and the required number of cytosolic siRNA molecules are correlated. The establishment of a dose-response relationship for intracellular siRNA/miRNA will aid substantially in the development and characterization of diverse novel delivery strategies including ligand-conjugated siRNA and exosomes.

Popular Abstract

RNA interference completely inactivate gene expression with 1500-2000 siRNAs

Cancer, among other genetic diseases, are caused by cells with malfunctioning genes that the body loses control of. Because our genes are expressed into a wide variety of protein structures, only one third can be treated with traditional medicine while the rest are practically undruggable. However, a new potentially revolutionary therapeutic approach called RNA interference (RNAi) can interfere with the genes, using short interfering RNAs (siRNAs). We found that between 1500-2000 of these siRNAs are enough to completely inactivate a gene.

The central dogma of molecular biology is how our genetic blueprint (e.g. our genes) are read and expressed into what ultimately becomes us. When a gene is expressed into a protein that i.e. gives us our eye color, the DNA of that gene is first copied into an mRNA before it is translated into a protein. RNA interference (RNAi), a new therapeutic method for treatment of genetic diseases, hijacks a machinery inside the cells that degrade the mRNA before it becomes protein, using short interfering RNAs (siRNAs). These are short sequences of synthetically genetic code that can be tailor-made to inactivate any gene of interest by simply composing them into sequences complementary to the gene’s mRNA sequence. Due to the recent discovery of RNAi, however, we only have limited knowledge about it. A current issue hampering the development of RNAi-drugs is the delivery inside the target cells. To improve knowledge about it, we set out to investigate the number of siRNAs that is necessary to inactivate a gene. We therefore developed a method that tracks individual cells over time and count the number of siRNAs causing inactivation of a certain gene.

We used a very sensitive fluorescent microscope that can record living cells and illuminate desired molecules that have been purposely labelled. To visualize the RNAi occurring, we labelled the siRNAs and the protein product of the gene that we wanted to inactivate. After recording the cells, we used various software that identified and followed every cell and measured the level of brightness glowing from the labelled molecules. These measurements were then translated into the number of siRNAs and correlated to how well they inactivated the protein.

The siRNA was delivered in endosomes, membrane-like bubbles, and was released into the cells at different occasions where it engaged with the RNAi machinery. Because of this, we wanted to begin measuring the inactivation of the target protein at that moment. Therefore, we developed an algorithm that could detect when these siRNA release events occurred. Before using it, we investigated if it was sensitive enough to detect the majority of cells having release events. We found that it indeed was sensitive enough, and could detected ~90% of the cells having a siRNA release. We then used the same algorithm to detect siRNA events in cells producing the labelled target protein, to investigate how many siRNAs that was needed to silence the gene. We found that approximately 1500-2000 siRNAs had to enter the cells and interact with the RNAi machinery for it to happen.

The next step will be to use this method to investigate other siRNA sequences targeting the same gene, and how the RNAi machinery functions in other cell-types. Other types of delivery strategies that can be used to deliver siRNA inside a cell will also be investigated, to improve knowledge about their efficacy as it is currently lacking.

Master’s Degree Project in Molecular Biology 30 credits 2018
Department of Biology, Lund University

Advisor: Anders Wittrup
Department of Clinical Sciences

• author: Hedlund, Hampus
• supervisor: Anders Wittrup
• organization: Degree Projects in Molecular Biology
• course: MOBM02 20181
• year: 2018
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 8962866
• date added to LUP: 2018-11-06 14:26:44
• date last changed: 2018-11-06 14:26:44