Chemical Research On SiRNA Druggability:Universal Base Modification And Nano-scaled Delivery System

RNAi (RNA interferencing) won Noble Prize in2006. siRNA is not only a fantastic molecular biology research tool, but also it’s a potential drug for human being. Recently, siRNA therapeutical application is popular in research institutes and companies. Billion of dollar has been invested into this field. Until now, none of siRNA dug received permission from FDA. The therapeutic use of synthetic siRNA is currently bumped by lack of efficient means of siRNA delivery, low stability in serum, and "off-target" effect due to low specificityIn this thesis, we tried to overcome some of the difficulties. The first part is the nucleo modification of5-nitroindole. Accumulating evidence shows that siRNA passenger strand can also assemble into RISC complex and mediate RNA interference, causing undesired off-target effect. To reduce this effect, the so called "universal base"5-nitroindole nucleotides was incorporated into siRNA passenger strand. Melting temperature (Tm) and circular dichroism (CD) spectrum measurements showed no significant changes compared to the unmodified duplex, indicating the forming of normal A-form conformation. Using dual luciferase reporter assay, we have further shown that5-nitroindole modification at position15of siRNA passenger strand drastically decreased RNAi (RNA interferencing) potency of this strand, while the potency of siRNA guide strand was not much affected. These results may provide a practical approach for reducing the off-target effect mediated by siRNA passenger strand. In the second part, we designed a new nano-scaled siRNA delivery system. Certain tissues, including liver, spleen, and some tumours, allow the passage of molecules up to1000nm in diameter, while in general, molecules larger than5nm in diameter do not readily cross the capillary endothelium. Nanoparticles constructed by minimally chemical modified siRNA are expected an ideal high-loading siRNA platform for RNAi therapeutically applications. Here, we described well-dispersed, size-controllable Archimedean Solid-like RNA nanoparticles (ASRN) with single siRNA sequence. It was proved that these orbicular particles are biofunctional. They can be digested by Dicer enzyme into21nt siRNA segments. The transfected siRNA nanoparticles showed extraordinary serum stability and excellent RNA interferencing effect (by dual-luciferase reporter assay in HEK293). These findings may contribute to both the design of nano-scaled siRNA containing complex and the controllable nucleic acid nanotechnology.By means of these two methods, we provide choices for the main concerns in siRNA therapeutical application.5-nitroindole modification can reduce the off-target effect by the sense strand. Construction of ASRN:1) siRNA serum stability improved;2) the nanoparticles are biofunctional;3) minimal modification of siRNA reduce the possibility of toxicity and immunostimulatory.