Study On The Chemical Synthesis Of Nucleic Acid And Synthesis, Evaluate The 4’ -C-hydroxymethyl Modified SiRNA

RNA Interference (RNAi) is one of the most important technological breakthroughs in modern Chemcalbiology. From its discovery in 1990, to 2006 Fire and Mello won the Nobel Prize in Physiology or Medicine for their work in it, RNA interference has experienced a extremely fast development period. Especially in the pharmaceutical field, The antisense and antigene approaches to chemotherapy could well revolutionise the treatment of cancer, certain viral infections and a number of other diseases in years to come, and more and more oligonucleotide sequences are presently undergoing advanced human clinical trials.These all will mean a great demand for the large-scale synthesis of oligonucleotides has become a matter of considerable importance and indeed of urgency. However, the shortcomings of tranditional approach for synthesis of oligonucleotides in solid phase are very obvious, such as high cost and low scale. Based on this point, this work made a study of two aspects in the synthesis of RNA:Firstly, The combined use of Phosphoramidite and H-phosphonate Approach in the solution phase synthesis of oligonucleotides. We utilized the Phosphoramidite Approach to synthesis of 5mer building blocks at first, and then assembled of the fully-protected 20 mer sequence through H-phosphonate Approach, with a ideal productivity. At last we removed all of the protection groups to obtain the 20 nt RNA sequence.Secondly, because some disadvantages of H-phosphonate Approach in the synthesis of RNA, we select the double cyanoethyl and borane base to protect hydrogen phosphate. This change can not only avoid the migration of TBDMS Group, but also can greatly ease the cycle route of condensation reaction.After the rapid development of RNAi technology, its defects have gradually emerged, such as off-target effects, easily be degraded in the living body, the construction of drug delivery system. We decide to synthesis 4’-C-hydroxymethyl modified siRNA to study its properties. Therefore, we firstly synthesized the 4’-C-hydroxymethyl modified phosphoramidite monomer, and then synthesis of modified nucleic acid with the phosphoramidite approaches in solid phase. At last, we evaluate the 4’-C-hydroxymethyl modified siRNA, including thermodynamic stability, retention of configuration and interference activity. We have found that some postions that modified by 4’-C-hydroxymethyl will siglificantly alter RNAi efficiency.