Light-activated Small Interfering RNAs

小干扰RNA(SiRNAs)是一种很有前途的基因沉默工具，可用于治疗多种病理疾病，如癌症、病毒感染、遗传病和自身免疫性疾病。这些短双链RNA由两个21-23个核苷酸链组成，其中一个正义链包含目标mRNA序列及其补体，即活性反义链。一旦进入细胞，RNA诱导的沉默复合体(RISC)将结合反义链，通过碱基对互补识别目标mRNA分子，最终酶降解该mRNA。通过20年前发现的这种机制(称为RNA干扰或RNAi)，siRNAs可以通过降解任何基因的mRNA来下调其表达，从而抑制后续蛋白质的功能。然而，需要取得新的进展来克服一系列限制，这将使siRNA发挥其作为广泛治疗药物的潜力。与大多数寡核苷酸药物类似，siRNA的一个主要限制是它们进入细胞。由于其相对较大的尺寸和负电荷，siRNA分子很难通过细胞膜运输。此外，在细胞内，它们也容易受到核酸酶的降解。另一个限制是siRNA特异性，因为它们在所有细胞类型和交付位置都是活跃的，如果需要仅针对特定类型的细胞或特定区域的细胞，这尤其是一个问题。为了克服这些问题，研究人员一直在对siRNA分子进行化学修饰。例如，保护siRNA免受核酸酶降解的一种方法是将磷酸二酯键转化为硫代磷酸，或修饰核糖的2‘-位，这也可以防止不必要的免疫反应或脱靶结合。另一种促进转运和细胞摄取的方法是将siRNA偶联到纳米载体上，如球形纳米颗粒。最近，几个研究小组开发了光激活RNA干扰技术，以实现对活动的时空控制。首先，为了阻止RISC对siRNA分子的访问，应用了不同类型的化学修饰。然后，在特定波长的辐射会触发分子的化学修饰部分的光裂解，这导致siRNA以活性形式释放，使其能够与RISC形成络合物并降解特定的mRNA。到目前为止，所有开发的光激活技术方法都存在几个缺点，包括泄漏关闭状态和在照明时仅部分激活。该项目旨在开发更可靠和更广泛适用的siRNA化学修饰，以解决光激活问题，这也可能具有改善递送和稳定性的额外好处。这些改进将有助于开发小干扰RNA作为一种治疗方法。该项目属于EPSRC化学生物学和生物化学以及生物材料和组织工程研究领域，是物理科学和医疗保健技术主题的一部分。

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.