Final Activity Report Summary - DESIGN OF NEW NUCLEO (Design of new nucleosides for DNA interactions and fluorescent studies)
This project is focus on the study of the effect of chemically modified RNAs in RNA interference. Specifically, the project wanted to address the role of the hydrogen bonding stabilisation and steric effects on RNA interference. We prepared several non-polar isosteres of natural nucleosides, these molecules are very close in size and shape than their natural counterparts but lack the polar groups involved in base pairing. These molecules are good probes to study the importance of hydrogen bonding interaction in biological systems. In this case we wanted to study this interaction in a gene regulation mechanism known as RNA interference. This process can knock down the expression of a specific protein by targeting the corresponding mRNA sequence. This process is triggered when a doubled stranded RNA (siRNA) reach the cytoplasm, then one of the strands known as guide is incorporated into a protein complex (RISC), which then cleaves the complementary sequence present in the mRNA.
In this project, we have incorporated several analogs to different positions of two different RNA sequences. After purification of the modified RNA strand they were annealed with the complementary sequence and the corresponding thermal stability was determined. The RNA interference experiments were carried out on HeLa cells using the dual-luciferase system. The results have shown that the hydrogen bonding stabilisation is not required at every position; moreover at position 7 of the guide strand the activity is high despite the low thermal stability. Therefore we have carried out selectivity studies at position 7 utilising mutated target sequences. The results of this set of experiments have shown that steric effects are critical in selectivity. This work has been published in Angewandte Chemie Int. Ed. 2006, 45, 4994-4997.
In this project, we have incorporated several analogs to different positions of two different RNA sequences. After purification of the modified RNA strand they were annealed with the complementary sequence and the corresponding thermal stability was determined. The RNA interference experiments were carried out on HeLa cells using the dual-luciferase system. The results have shown that the hydrogen bonding stabilisation is not required at every position; moreover at position 7 of the guide strand the activity is high despite the low thermal stability. Therefore we have carried out selectivity studies at position 7 utilising mutated target sequences. The results of this set of experiments have shown that steric effects are critical in selectivity. This work has been published in Angewandte Chemie Int. Ed. 2006, 45, 4994-4997.