Light-activated Small Interfering RNAs

Small interfering RNAs (siRNAs) are a promising gene silencing tool for the treatment of a variety of pathological conditions, such as cancer, viral infections, genetic diseases and autoimmune disorders. These short-double stranded RNAs are composed of two 21-23 nucleotide strands, with a sense strand containing the target mRNA sequence and its complement, the active antisense strand. Once inside the cell, the RNA-induced silencing complex (RISC) will bind the antisense strand and identify the target mRNA molecule through base-pair complementarity, ultimately degrading this mRNA enzymatically. Through this mechanism, discovered 20 years ago (named RNA interference or RNAi), siRNAs can potentially downregulate the expression of any gene by degrading its mRNA, hence inhibiting the function of the subsequent protein. However, new advances need to be made to overcome a series of limitations, which would then allow siRNAs to achieve their potential as a broad therapeutic. Similar to most oligonucleotide drugs, a main limitation for siRNA is their delivery into cells. Due to its relatively large size and negative charge, siRNA molecules are difficult to transport across the cell membrane. Additionally, within the cell they are also susceptible to nuclease degradation. Another limitation is siRNA specificity, as they will be active in all cell types and locations they are delivered, which is particularly a problem if the need is to only target a certain type of cells or cells in a specific area. To overcome these problems, researchers have been introducing chemical modifications of the siRNA molecule. For instance, one way to protect siRNA from nuclease degradation is the conversion of phosphodiester bonds into phosphorothioates, or the modification of the 2'-position of ribose, which can also prevent undesired immune responses or off-target binding. Another reported method to facilitate transport and cellular uptake is the coupling of siRNA to nanocarriers, such as spherical nanoparticles. Recently, light-activated RNA interference technologies have been developed by several research groups, to achieve spatiotemporal control of activity. First, different kinds of chemical modifications are applied in order to block the access to the siRNA molecule from RISC. Then, irradiation at a certain wavelength triggers a photocleavage of the chemically modified portion of the molecule, which leads to the release of siRNA in an active form, leaving it able to form the complex with RISC and degrade a specific mRNA. To date, all the developed methods of light-activated technology present several downsides, including a leaky OFF state and only partial activation upon illumination. This project aims to develop more reliable and broadly applicable chemical modifications of siRNA to address the issues with light-activation, which may also have the added benefit of improving delivery and stability. These improvements will aid in the development of small interfering RNA as a therapeutic. This project falls within the EPSRC Chemical Biology and Biological Chemistry and Biomaterials and Tissue Engineering research areas, part of the Physical Sciences and Healthcare Technologies themes.